Design, synthesis and biological evaluation of amide-pyridine derivatives as novel dual-target (SE, CYP51)antifungal inhibitors

Accepted Manuscript

Design, synthesis and biological evaluation of amide-pyridine derivatives as

novel dual-target (SE, CYP51) antifungal inhibitors

Bin Sun, Yue Dong, Kang Lei, Jian Wang, Liyu Zhao, Min Liu

PII:

S0968-0896(18)32111-4

https://doi.org/10.1016/j.bmc.2019.02.009

BMC 14745

DOI:

Reference:

Bioorganic & Medicinal Chemistry

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Revised Date:

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18 December 2018

28 January 2019

2 February 2019

Please cite this article as: Sun, B., Dong, Y., Lei, K., Wang, J., Zhao, L., Liu, M., Design, synthesis and biological

evaluation of amide-pyridine derivatives as novel dual-target (SE, CYP51) antifungal inhibitors, Bioorganic &

Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.02.009

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Design, synthesis and biological evaluation of amide-pyridine

derivatives as novel dual-target (SE, CYP51) antifungal

inhibitors

Bin Sun ^a^, Yue Dong ^a, Kang Lei ^a, Jian Wang ^b, Liyu Zhao ^b, Min Liu ^a



^aInstitute of BioPharmaceutical Research, Liaocheng University, 1 Hunan Road, Liaocheng 252000, PR China

^bKey Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, School of Pharmaceutical

Engineering, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang 110016, PR

China

Abstract:

Based on the analysis of the squalene cyclooxygenase (SE) and 14α-demethylase

(CYP51) inhibitors pharmacophore feature and the dual-target active sites, a series of

compounds with amide-pyridine scaffolds have been designed and synthesized to treat

the increasing incidence of drug-resistant fungal infections. In vitro evaluation

showed that these compounds have a certain degree of antifungal activity. The most

potent compounds 11a, 11b with MIC values in the range of 0.125-2 μg/ml had a

broad-spectrum antifungal activity and exhibited excellent inhibitory activity against

drug-resistant pathogenic fungi. Preliminary mechanism studies revealed that the

compound 11b might play an antifungal role by inhibiting the activity of SE and

CYP51. Notably compounds did not show the genotoxicity through plasmid binding

assay. Finally, this study of molecular docking, ADME/T prediction and the

construction of 3D QSAR model were performed. These results can point out the

direction for further optimization of the lead compound.

Keywords: Dual-target; Amide-pyridine compounds; Antifungal activity; Molecular

docking; 3D QSAR model.

1. Introduction

Fungal infection is a common disease, which can be divided into superficial and

deep fungal infections depending on the location of the infection.¹In recent years, the

incidence of fungal diseases has become more and more frequent with the abuse of

clinical broad-spectrum antimicrobial agents, chemotherapy drugs and immuno

-suppressive agents.^{2, 3}At the same time, the emergence of drug-resistant fungal

diseases has also increased significantly, and it is very difficult to find the proper

method of treatment. In addition, the update speed of antifungal drugs cannot meet the

increasing demand for disease treatment.⁴Therefore, it is becoming more and more

important to discover new antifungal inhibitors and solve the problem of fungal

resistance.

At present, the design and development of antifungal drugs is mainly performed by

interfering with the biosynthesis of glucan and ergosterol.⁵The inhibitors affecting the

glucan synthesis are mainly some antibiotics, which show excellent clinical

characteristics, but they also have the defects such as inability to attain oral

administration, difficulty in synthesis and high price.^6-8While the most of ergosterol

synthase inhibitors don't have these problems, they can interact with the specific

targets to achieve the purpose of treatment. The mechanism of ergosterol synthesis

was further analyzed, and the rate-limiting enzymes squalene cyclooxygenase (SE)

and 14α-demethylase (CYP51) played an important role in the process.^9-11If the

activity of the rate-limiting enzymes is inhibited, ergosterol synthesis would be

blocked, and the upstream components accumulate in the cells. Finally, it can lead to

the fungal cell death.^{12, 13}These single-target enzyme inhibitors, such as acrylamines,

thioguanides and azoles compounds, have been widely and effectively applied in

clinic.^14-17However, the phenomenon of toxic side effects and drug resistance is

constantly emerging with the long-term use of these inhibitors. ¹⁸Dual-target drugs or

multi-target drugs can avoid these disadvantages.^{19, 20}

At present, the squalene epoxidase (SE) and 14α-demethylase (CYP51) inhibitors

are widely used in the treatment of drug-resistant fungal diseases by combination

therapy, and they can show good synergy and additional effects in clinic.^21-23In

addition, those inhibitors have similar spatial structure in our previously report.¹¹

Based on this principle, we hope to construct the novel antifungal inhibitors with

dual-target properties by analyzing the molecular structural characteristics of squalene

epoxidase (SE) and 14α-demethylase (CYP51) inhibitors. In the study, the

fragment-based drug discovery (FBDD) approach was applied to design a series of

novel dual-target inhibitors,²⁴and these compounds were synthesized. The antifungal

activity was evaluated using biochemical and cell-based assays. This study will

provide a new direction for the design of new antifungal inhibitors.

2. Results and discussion

2.1 The design of dual-target antifungal agents

In order to design the new skeleton of dual-target antifungal inhibitors, the

pharmacophore feature models and molecular docking of squalene epoxidase (SE)

and 14α-demethylase (CYP51) inhibitors, which were constructed in our previously

report, were analyzed.^{11, 25, 26}

At first, the pharmacophore model of SE inhibitors consists of three hydrophobic

groups (HHH), while the pharmacophore model of CYP51 inhibitors comprises three

hydrophobic groups and one hydrogen acceptor (HHHA) (Figure 1. A, B). These

features of pharmacophore models contain the common hydrophobic (H) features in

the orientation, composition and vector directions, and have the similar spatial

distribution. The essential main chain fragments of SE and CYP51 inhibitors are

composed of aromatic groups, and they could be appropriately matched to the

hydrophobic groups, respectively. While the nitrogen-containing fragments of CYP51

inhibitors were positioned in hydrogen bond acceptor. At the same time, these

fragments were connected by the flexible branched chains.

Figure 1. (A) The pharmacophore model of SE inhibitors consisting of three hydrophobic groups

(H); (A₁-A₂) The binding mode of SE inhibitors in the active site of SE. (B) The pharmacophore

model of CYP51 inhibitors consisting of three hydrophobic groups (H) and a hydrogen bond

acceptor (A); (B₁-B₂) The binding mode of CYP51 inhibitors in the active site of CYP51.

Distances between the features are expressed in Å, with a tolerance sphere of radius ±0.9 Å.

From the results of molecular docking (Figure 1. A₁-B₂), it was observed that the

active sites of SE and CYP51 are mainly composed of hydrophobic amino acid

residues. Therefore, both SE inhibitors and CYP51 inhibitors exhibit hydrophobic

properties; they could produce the proper interaction with the respective active sites.

At the same time, the binding modes of SE and CYP51 inhibitors with active sites are

also similar, the SE and CYP51 inhibitors containing the essential main chain

fragments were combined with their respective active pockets of target enzymes

through the hydrophobic interactions; The phenyl fragment of SE inhibitor can form

the hydrophobic interactions with Pro307 and Met333 at the active sites of SE. While

the pyridine fragment of CYP51 inhibitor also plays an important role, which can

form the key coordination bond with heme in the active cavity of CYP51.

Figure 2. (A) The superposition pattern of SE inhibitor and CYP51 inhibitor. (B) Design of amide

pyridine derivatives as novel dual-target antifungal inhibitors.

Based on the detailed pharmacophore feature models and inhibitor-enzyme

interaction models, we can know that the main chain fragments, phenyl fragment and

nitrogen-containing fragment as the core regions play an important role in their

respective inhibitory activities (Figure 2). The design strategy for dual target

inhibitors was to retain the core regions, and replace these regions fragments with

other active groups. The biphenyl group and arylpyrrole group were chosen as the

main chain fragments. The nitrogen-containing fragment can form the coordinate

bond with the heme group in the active site of CYP51. Therefore, the pyridyl group

was chosen to replace the nitrogen-containing fragments of the CYP51 inhibitor and

the phenyl fragment of the SE inhibitor. At the same time, the side-chain region (R)

was investigated by using different sizes of spatial structural fragments. Finally, the

different structural regions were linked through flexible branched chains.

2.2 Chemistry

The synthetic routes of the key intermediates (3) are illustrated in Scheme 1.¹⁰

Commercially available starting material 2-nitrobenzaldehyde (1) was treated with

ferrous sulfate and ammonia in the presence of N, N-dimethylformamide to provide

the intermediate 2-aminobenzaldehyde (2). Subsequently, the 2-aminobenzaldehyde

was treated with the methyl pyruvate in the alkaline conditions to give the key

intermediate 2-quinolinecarboxylic acid (3).

Scheme 1. Reagents and conditions: (a) FeSO₄.7H₂O, NH₃.H₂O, reflux, 0.5 h; (b) Methyl

pyruvate, KOH, 80°C, 3 h.

The target compounds 8a-h, 11a-h were prepared according to the synthetic route

outlined in Scheme 2. First, L-Glycine, L-Alanine, L-Valine and L-Phenylglycine

(4a-d) were dissolved in ethanol and refluxed with SOCl₂for 3 h, and the amino acid

ethyl ester hydrochloride (5a-d) were obtained, respectively. ²⁷Next, these amino acid

ethyl ester hydrochlorides were separately treated with the key intermediate

2-naphthoic acid and 2-quinolinecarboxylic acid (3) in the presence of condensing

agent to give the required products (6a-d, 9a-d). Subsequently, these compounds were

hydrolyzed with 2N sodium hydroxide solution to obtain the corresponding organic

acids (7a-d_,10a-d). Finally, the target compounds 8a-h and 11a-h were synthesized

from organic acids and aminomethylpyridine via amidation reaction.

Scheme 2. Reagents and conditions: (c) Alcohol reagent, SOCl₂, reﬂux, 2-3 h; (d) EDCI, HOBt,

DIEA, 2-Naphthoic acid or 2-Quinolinecarboxylic acid, 80°C,7 h; (e) 2N NaOH solution, MeOH,

60 °C, 5 h; (f) PyBOP, DIEA, Aminomethylpyridine, rt, 7 h.

2.3 In vitro antifungal activity

In order to evaluate the antifungal activities of target compounds against the

different fungal pathogens, the antifungal experiments in vitro were performed

according to the protocols from the NCCLS.^{10, 27}Fluconazole (FLC) and Terbinafine

were selected as reference drugs. The broth microdilution method was used to

determine the minimum inhibitory concentrations (MICs) of the target compounds in

96-well plates.

In vitro antifungal activities of the target compounds are listed in Table 1. It can be

seen that most of the target compounds exhibited moderate to high antifungal

properties with broad spectrums of activity, which suggests the rationality of the

antifungal inhibitor design. In particular, compounds (8a-c, 8e-f, 11a-b and 11e-f)

exhibited better or similar antifungal activity than reference drugs FLC and

Terbinafine with MIC values in the range of 0.125-2 μg/ml by inhibiting pathogenic

Candida fungi (C. albicans, C. glabrata, C. krusei and C. tropicalis). In addition,

these compounds showed a tendency to gradually decrease in activity in inhibiting

Candida with the increase of the spatial structure of side chain groups. The reason

may be that the substituted groups can affect the binding of compounds to target

enzymes. At the same time, the antifungal activity of the 4-pyridyl group is slightly

increased compared to the 3-pyridyl group. It is noteworthy that some compounds 8b,

11a-b are also shown the activity against Aspergillus fumigatus, and they deserve our

further study.

Table 1. In vitro antifungal activities of the target compounds (MIC, μg/ml).

Compd

R

R₁

MIC, µg/ml

C.alb.

0.25

2

C.gla.

C.kru.

1

C.tro.

0.5

0.25

2

A.fum

2

H

4-picolinylamine

3-picolinylamine

4-picolinylamine

3-picolinylamine

̶

0.25

1

8a

Methyl

Isopropyl

Phenyl

H

0.5

4

2

8b

0.5

8

8c

4

>16

0.5

2

>16

4

8d

1

4

2

8e

Methyl

Isopropyl

Phenyl

H

0.5

8

1

0.5

4

8f

8g

2

>16

2

>16

0.25

0.125

0.5

2

>16

0.5

0.125

2

8

8h

0.5

0.25

0.5

4

0.125

0.25

4

11a

Methyl

Isopropyl

Phenyl

H

1

11b

>16

2

11c

>16

2

>16

1

11d

0.5

1

0.5

2

11e

Methyl

Isopropyl

Phenyl

̶

0.25

0.5

4

0. 5

2

8

11f

0.5

8

1

4

11g

>16

0.5

2

>16

2

11h

0.5

0.25

1

2

Fluconazole

Terbinafine

̶

1

2

Abbreviations: C. alb., Candida albicans (ATCC 10231); C. gla., Candida glabrata (ATCC 0001); C. kru.,

Candida krusei (ATCC 6258); C. tro., Candida tropicalis (ATCC 1369); A. fum., Aspergillus fumigatus

(KM8001).

2.4. In vitro antifungal activity against Fluconazole-resistant strains of C. alb

Fluconazole has shown good efficacy in the treatment of fungal diseases, especially

Candida albicans. However, the problem of drug-resistance has become more and

more frequent with the widespread abuse of Fluconazole. Based on the analysis of

antifungal experiments in vitro, the most potent compounds 11a, 11b were selected to

detect the inhibitory activity against fluconazole-resistant strains of C.alb. (Strain 100

and strain 103). The results are shown in Table 2. Compound 11a and 11b

demonstrated excellent inhibitory effect on fluconazole-resistant strains of Candida

albicans with MIC values in the range of 0.5-2 ug/ml.

Table 2. In vitro antifungal activities of the target compounds (MIC, μg/ml) ^a.

Compd

MIC, μg /ml

C.alb. Strain100

C.alb. Strain103

＞16

8

＞16

Fluconazole

Terbinafine

11a

4

1

2

0.5

11b

^aAbbreviations: C. alb., Candida albicans; strain 100, ﬂuconazole-resistant strains of Candida albicans; strain

103, ﬂuconazole-resistant strains of Candida albicans; Strain 100 and strain 103 were provided by The Second

Military Medical University.

2.5 Morphological analysis of C. alb. Strains with different concentrations of

drugs

In order to further understand the effects of different compounds on the

morphology of pathogenic fungi cells, the morphology of Candida albicans was

observed by polarizing microscopy at different drug concentrations (Table 3).

In the blank control group, Candida albicans exhibited the proliferation

phenomenon, and the cell morphology showed elliptical shape, a large number of

Candida cells appeared spores and cell germination; some fungal cells elongated and

formed pseudo hyphae. In the medium/high concentration (250 nM/ml and 25 nM/ml)

groups, the results showed that the target compounds (11a, 11b) inhibited the growth

of Candida cells even better than the positive control drugs (Fluconazole and

Terbinafine). They not only effectively decreased the division but also killed the

existing Candida cells. At this time, the volume of Candida albicans decreased, and

the proportion of Candida cells in the division phase was significantly lower than that

in the blank control group. In the low concentration (2.5 nM/ml) group, the efficacy

of the positive control drugs reached the critical concentration and could not inhibit

the growth of the fungus. The target compounds can still show the inhibition effect on

Candida cells, and some cells even have the fracture phenomenon.

Table 3. Polarizing microscopy results of target compounds and the positive control drugs against

Candida albicans in different concentrations.

2.6 Analysis of antifungal mechanisms

In order to study the antifungal mechanism of target compounds, the components of

C. albicans cells were extracted.^{10, 27}We analyzed the effects of target compound 11b

on the sterol composition of Candida albicans. Fluconazole and Terbinafine were

used as the reference drug; the analysis result is shown in Figure 3 and Table 4.

Figure 3. Analysis of sterol composition in untreated group (A), FLC-treated group (B),

Terbinafine-treated group (C), compound 11b-treated group of C. albicans.

Table 4. Analysis of composition in C. albicans.

Contents.

% of total sterols (Candida albicans)

Control

64.20

8.30

Fluconazole(FLC)

Terbinafine

10.61

Compound 11b

11.31

39.46

49.23

-

9.02

27.45

50.08

13.45

-

Ergosterol

Eburicol

21.87

10.99

-

22.44

Lanosterol

Squalene

37.81

16.51

-

7.27

Unidentified

In the untreated group, ergosterol was determined as the main component, which

accounted for 64.2% of the extract contents, eburicol and lanosterol comprised only

19.29% of the total contents. In the positive control groups, the C. albicans was treated

with Fluconazole and Terbinafine for 16 h, and the content of ergosterol was reduced to

11.31% and 10.61%, respectively. In the fluconazole treatment group, the contents of

lanosterol and eburicol were significantly increased in the fluconazole treatment group,

but squalene was not observed. These data indicate the Fluconazole plays the antifungal

role by inhibiting CYP51. While the squalene content was observed to show a

significant increase in the terbinafine treatment group, the contents of lanosterol and

eburicol also slightly increased, which indicate Terbinafine exerts the mainly antifungal

activity by inhibiting squalene cyclooxygenase. In the compound 11b treatment group,

the content of ergosterol decreased to 9.02%, while the content of lanosterol and

eburicol increased to 50.02% and 27.45% of the total contents, respectively. It is worth

noting the squalene content was also observed, which reveal the compound 11b can

affect both squalene epoxidase and CYP51 activity at the same time.

2.7 Heme Binding Studies of target compound 11b

Heme is an important component of CYP51 active site, and the binding of target

compound 11b to heme can indirectly reflect the bind ability of the target compound

to CYP51. Thereby, the heme binding properties of compound 11b was evaluated via

multiple spectral methods. ²⁸

Figure 4. (A) UV-vis spectra of heme with compound 11b and Fluconazole; (B)

Fluorescence spectra of heme in the absence and presence of compound 11b

.

The UV-vis and Fluorescence spectrum were employed to validate the

combination of compound 11b with heme. The results were depicted in Figure

4A, 4B. In the UV-vis spectra, the absorption peaks of heme were found at

383.00 nm and 608.00 nm, and the absorption peaks of target compound 11b

and Fluconazole did not exist in this region. When Fluconazole and target

compound 11b were added to heme solution, the absorption peak of heme was

shifted upward. These facts prove that the target compound 11b has the same

action mechanism with Fluconazole, and they can cause the changes in the

ultraviolet absorption spectrum of heme. In the Fluorescence spectra, it reveals

that heme displayed strong fluorescence emission at 553 nm under the

excitation of 550 nm. The typical fluorescence emission of heme quenches

gradually shifted down with the increasing concentration of target compound

11b (0.0–50.0nm/ml) in solution. These facts evidence heme can interact with

the target compound 11b. If the fluorescence intensity is proportional to the

concentration of the heme, the relationship between the fluorescence intensity

and the drug concentration can often be expressed as

log(F0-F)/F=logKa+nlog[Q]

while [Q] is the free concentration of a drug. Ka and n are the binding

constant and the number of binding sites, respectively, which can be obtained

from the linear plot of log (F0-F)/F versus log [Q].^{29, 30}After calculation, the

relevant data were obtained (n=1, Ka=199

10⁵M^-1), which indicate that

compound 11b can form the strong interaction with heme at a specific binding

site.

2.8 Molecular docking

Molecular docking was performed to investigate the interaction between the target

compound 11b and the dual-target enzymes (CYP51, SE), and it can provide the

guide for further structure-based drug optimization. The target compound 11b was

docked into the active site by using the CDOCKER program in Discovery Studio 3.0.

Images depicting the proposed binding modes were generated using PyMOL, which

are shown in Figure 6.

In the docking model, compound 11b was docked in SE and CYP51 enzyme,

respectively. From this result, it can be seen that the active chamber of SE exhibited a

long column shape, compound 11b can properly match the active chamber of SE, and

the various groups of the compound can interact with the key amino acid residues

around the active chamber. The pyridine group of the compound 11b can form

pi-alkylation with Pro38, and the quinoline group can form pi-alkylation with Val12

and Ile13 (Figure 6 A₁-A₃). In addition, the flexible branched chain containing amide

bond groups can produce the hydrogen bonding interaction with Ile41 and Gly198,

respectively. At the same time, compound 11b can also be docked into the active

cavity of the target enzyme CYP51. The pyridine group of compound 11b occupied

the substrate-binding site of the CYP51 enzyme, and formed the coordinate bond with

the heme group of CYP51 (The coordinate bond length is 2.707 Å). The quinoline

group of the compound was surrounded by hydrophobic residues (Leu 121, Pro 230,

Phe233, Ile379, Phe380), and formed the hydrophobic interactions with the key

residues Pro230 and Phe233, respectively (Figure 6 B₁-B₃).

Figure 5. Docking models of representative compound 11b. (A₁) The molecular docking

modeling of compound 11b with SE. (A₂) 3D model of the interaction between compound 11b and

SE binding site. (A₃) 2D molecular docking modeling of compound 11b with SE. (B₁) The

molecular docking modeling of compound 11b with CYP51. (B₂) 3D model of the interaction

between compound 11b and CYP51 binding site. (B₃) 2D molecular docking modeling of

compound 11b with CYP51.

2.9. Theoretical evaluation of ADME/T properties

The properties of ADMET include the absorption, distribution, metabolism,

excretion and toxicity of drugs in the human body. It is very important for improving

the success rate of drug research and reducing the waste of funds. In order to further

study the drug production of target compounds, the ADMET values of positive control

drug and compounds 11a, 11b were predicted and analyzed.

The two analogous 95% and 99% confidence ellipses for the blood-brain barrier

(BBB) penetration and human intestinal absorption (HIA) models are shown in the 2D

diagram of ADMET_AlogP98 vs ADMET_PSA_2D, respectively (Figure. 7). All the

target compounds and the control drug (Fluconazole) were distributed in the 99%

confidence region for BBB penetration and HIA, and Terbinafine was excluded from

the 95% confidence ellipses of HIA (Absorption-95), which indicate that the ADMET

values of target compounds exhibit appropriate reliability.

The detailed results for the ADMET properties are shown in Table 5. When the

value is less than 5, it indicates that the compounds' ADMET located in the reasonable

range. The range of compounds' BBB is -1.134-1.442, which indicate compounds 11a

and 11b exhibit low BBB penetration; these compounds can increase the drug action

time by inhibiting CYP2D6, compound 11a, 11b even have smaller hepatotoxicity

than Fluconazole and Terbinafine. In addition, the Alog P and solubility can represent

the compounds' hydrophilicity, which can directly affect the compounds' absorption

and penetration. The range of compounds' Alog P (0.75-5.335) and solubility (-6.128

-1.976) are also located in the allowed range. Likewise, the PSA parameter can

directly reflect the drug bioavailability. The PSA values of the compounds are all less

than 140, which indicate that the compounds retain the reasonable property of

bioavailability. Therefore, these compounds can be used as good starting point for

further developing and designing new derivatives.

̶

Figure 6. Plot of PSA versus Alog P for the candidate compounds. Abbreviations: ADME/T,

absorption, distribution, metabolism, excretion and toxicity; Alog P, the logarithm of the partition

coefficient between octanol and water; PSA, polar surface area; 2D, two-dimensional; BBB, blood

brain barrier.

Table 5. In silico ADME/T prediction of the potential compounds11a and 11b compared

with the Terbinafine and Fluconazole

ADME/T parameters

ADME/T-BBB

11a

11b

Terbinafine

Fluconazole

-0.96

-1.109

1.442

-1.134

-2.588

0.847

-3.083

1.336

-6.125

-0.378

17.1148

5.335

-1.976

11.222

-13.977

0.750

ADMET-Solubility

ADMET- Hepatotoxicity

ADMET-CYP2D6

AlogP98

-14.6736

1.145

-15.7927

1.629

82.744

3.352

76.556

PSA

2.10 3D QSAR model

The 3D QSAR model is a regression model based on the stereoscopic and

electrostatic fields of small molecules. It can be used to predict the activity of small

unknown ligands and observe the beneficial and unfavorable interactions between

receptors and ligands. Therefore, it is very important to construct the 3D QSAR

model of target compounds.

In the study, each molecule with lowest energy conformation was superposed by

the reference molecule (11b), all sixteen compounds were selected based on the

definite -log MIC values. Diverse Molecules method in Discovery Studio 3.5 was

utilized to choose training set and test set. The 3D QSAR model was constructed,

which was shown in Figure 8.

Figure 7. Molecular overlap of target compounds and the linear regression of GridBased

TempModel vs. arNCTRlogRBA.

The 3D QSAR model was acceptable with the correlation coefficient r²=0.884.

Molecules aligned with the iso-surfaces of the model coefficients on electrostatic

potential grids (Figure. 9 A-left) and Van der Waals grids (Figure. 9 B-right) were

listed. Electrostatic map indicated regions where electron-withdrawing (red) or

electron-donating (blue) groups would increase activity, while steric map indicated

areas where steric bulk would increase (green) or decrease (yellow) activity.

According to the analysis of maps, a promising orientation is that the upper part

region of main chain group may introduce some electron-withdrawing groups, and the

lower part region may make aromatic ring larger with electron-donating substitutes.

While the intermediate region of the side-chain groups should be properly introduced

into the electron-withdrawing group. At the same time, the steric hindrance of region

should also be reduced to enhance the binding ability of target compounds to dual

target enzyme. It is noteworthy that the pyridine group region has reached a relatively

perfect situation, which asked for a group with appropriate size and slighter electronic

influence. Through the analysis of the 3D-QSAD results, the optimization of target

compound is further indicated the direction.

Figure 8. 3D-QSAR of target compounds. (A) In the maps red contours mean

electron-withdrawing group is expected to increase activity while blue contours mean

electron-donating group is better. (B) Green areas mean steric bulk is better while yellow areas

mean small groups are helpful.

3. Conclusions

At present, the phenomenon of toxic side effects and the drug resistance of

pathogenic fungi are becoming more and more frequent. However, there is still no

effective treatment. The dual-target or multi-target drug therapy can adjust many links

of the disease network system at the same time, so it is not easy to produce drug

resistance and has good clinical effect. In this study, we first analyzed the

pharmacophore and molecular docking characteristics of squalene cyclooxygenase

(SE) and 14a-demethylase (CYP51) inhibitors, a series of amide-pyridine derivatives

were designed and synthesized by the method of fragment-based drug discovery

(FBDD). All the compounds demonstrated appropriate antifungal activities.

Compounds (8a-c, 8e-f, 11a-b, 11e-f) showed the antifungal with MIC in the range of

0.125-8 μg/ml toward different pathogenic fungi, especially compounds 11b showed

excellent antifungal activity and significant inhibition to drug-resistant fungi. The

analysis of fungal cell components and spectrum showed that the target compound

11b possessed simultaneously the inhibitory activity against SE and CYP51. At the

same time, these compounds did not show the genotoxicity through plasmid binding

assay. In addition, the molecular docking and prediction of ADME/T properties were

performed, respectively. The compound 11b can occupy the active sites of SE and

CYP51, and form the interactions with the key amino acid residues. At the same time,

the compounds 11a, 11b also demonstrate proper ADME/T properties, which suggest

that these compounds have good drug-forming properties. Finally, the construction of

the 3D QSAR model can guide the subsequent optimization and modification of the

amide-pyridine derivatives. In summary, these results demonstrate that these target

compounds have the potential to be developed as the lead compound.

Experimental section

4.1 Chemistry

All commercial reagents and solvents were purchased without additional

purification. The reaction progression was detected by thin layer chromatography

(TLC), it was performed on Silica Gel 60 F254 plates (Jiangyou, Yantai). Column

chromatography was run on silica gel (200–300 mesh) from Qingdao Ocean

Chemicals (Qingdao, Shandong, China). The melting points of all compounds were

determined with a Büchi Melting Point B-540 melting point apparatus (Büchi

Labortechnik, Flawil, Switzerland) and were uncorrected. The mass spectra (MS)

were determined in ESI mode on an Agilent 1200 LC-MS (Agilent, Palo Alto, CA,

USA). Nuclear magnetic resonance (1H-NMR and 13C-NMR) spectra were recorded

on a Bruker 400 MHz NMR spectrometer using TMS as an internal standard. The

chemical shifts were reported in parts per million (ppm) and the coupling constants (J)

were reported in Hertz (Hz). Peak multiplicities were expressed as follows: s, singlet;

d, doublet; t, triplet; q, quartet; dd, doublet of doublet; dt, doublet of triplet; td, triplet

of doublet; ddd, doublet of doublet of doublet; m, multiplet; br, broad.

4.2 General procedure for the synthesis of 2-aminobenzaldehyde (2)

2-nitrobenzaldehyde (7.6g, 0.05mol) and FeSO₄.7H₂O (139g, 0.5 mol) were added

to the weak acid aqueous solution, the mixture was stirred and heated at reflux for 1 h.

After that, 152ml (0.95mol) 25% ammonia solution was added in batches, the solution

was heated at reflux for 40 minutes. The reaction mixture was extracted with ethyl

acetate and brine, and the organic phase was dried over Na₂SO₄overnight, and the

solvent was removed under vacuum. The resulting solid was dried to give the desired

compound.

4.3 General procedure for the synthesis of quinoline-2-carboxylic acid (3)

2-aminobenzaldehyde (12.1 g, 0.1mol) and methyl pyruvate（12.3 g, 0.12 mol)

were added to the aqueous solution (100 ml), and the reaction mixture was stirred for

2 h at room temperature. Subsequently, the 10% potassium hydroxide aqueous

solution (80 ml) is continuously added to the reaction solution. The solution was

heated to 80 °C for 6 h, and then cooled to room temperature. The solution was

adjusted to pH =1-3 with 2 N HCl. The white solid precipitate was collected by

filtration and dried to give the desired compound.

4.4 General procedure for the synthesis of L-amino acid ethyl ester

hydrochlorides (5a-d)

L-amino acids (Glycine, Alanine, Valine, Phenylglycine) (1 equiv.) were dissolved

in ethanol solution, respectively; Thionyl chloride (3 equiv.) was added dropwise at

0 °C using a salted ice bath. The mixture was heated at reflux for 3-6 h. Then, the

reaction mixture was concentrated under reduced pressure to give the white solid

4a-d.

4.5 General procedure for the synthesis of compounds (6a–d, 9a–d)

The EDCI (1.1 equiv) and HOBt (1.1 equiv) were added to a solution of

appropriate organic acids (2-naphthoic acid, 2-quinolinecarboxylic acid and

benzofuran-2-carboxylic acid, 1 equiv) in anhydrous DMF, respectively. The mixture

was stirred at room temperature for 2 h. Then, L-amino acid ethyl ester 9a–d (1.1

equiv) and DIEA (4 equiv) were added, and the mixture was heated at 75 °C for 7 h.

The reaction mixture was poured into ice water, and extracted with ethyl acetate, and

the organic phase was dried over Na₂SO₄overnight. Finally, the desired compounds

were obtained by vacuum distillation.

4.6 General procedure for the synthesis of compounds (7a–d, 10a–d)

The compounds (10a–d, 14a–d, 17, 7.2 mmol) were dissolved in 15 ml methanol,

the solution of 2N sodium hydroxide 30 ml was added. Then, the reaction mixture

was stirred at 60 °C for 7 h. The reaction process was monitored by TLC analysis.

After the completion of reaction, the methanol was removed by rotary evaporation,

pH was adjusted to 2-3 by 2N hydrochloric acid, and white solid was filtered and

dried to give the desired compounds.

4.7 General procedure for the synthesis of compounds (8a–h, 11a–h)

The PyBOP (1.1 equiv) and key intermediate (12a–d, 15a–d, 18, 1 equiv) were

added into DMF, respectively. The mixture was stirred at room temperature for 2 h,

then the aminomethyl (1.1 equiv) and DIEA (4 equiv) were added, and the mixture

was heated at 80 °C for 7 h. The reaction mixture was poured into ice water, and

extracted with ethyl acetate, the organic phase was dried over Na₂SO₄overnight, and

the solvent was removed under vacuum. The resulting solid was dried to give the

desired compounds. The product was purified by flash column chromatography.

4.8.1 (R)-N-(2-oxo-2-((pyridin-4-ylmethyl)amino)ethyl)-2-naphthamide(8a)

1

Light white solid; yield: 64.2%; mp: 110.4–121.5 ℃. H NMR (400 MHz,

DMSO-d₆) δ 9.03 (t, J = 5.9 Hz, 1H), 8.67 – 8.43 (m, 4H), 8.13 – 7.96 (m, 4H), 7.77 –

7.58 (m, 3H), 7.39 (dd, J = 7.7, 4.9 Hz, 1H), 4.39 (d, J = 6.0 Hz, 2H), 4.02 (d, J = 5.9

13

Hz, 2H). C NMR (101 MHz, DMSO-d₆) δ169.71, 167.04, 149.12, 148.44, 135.40,

134.63, 132.54, 131.79, 129.30, 128.25, 128.20, 128.10, 128.06, 127.19, 124.71,

123.82, 43.36, 40.23. ESI-MS m/z: 320.0 [M+H]⁺; 342.1 [M+Na]⁺; 318.0 [M-H]^-.

HRMS calcd for C₁₉H₁₇N₃O₂, [M+H]⁺, 320.13208; found 320.13956.

4.8.2 (R)-N-(1-oxo-1-((pyridin-4-ylmethyl)amino)propan-2-yl)-2-naphthamide(8b)

1

Light white solid; yield: 68.3%; mp: 115.9–126.7℃. H NMR (400 MHz,

DMSO-d₆) δ 8.78 (d, J = 7.0 Hz, 1H), 8.67 – 8.41 (m, 4H), 8.16 – 7.90 (m, 4H), 7.70

– 7.55 (m, 2H), 7.29 (d, J = 5.7 Hz, 2H), 4.59 (p, J = 7.2 Hz, 1H), 4.35 (d, J = 6.0 Hz,

2H), 1.46 (d, J = 7.2 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 173.33, 166.82,

149.85, 149.08, 134.63, 132.52, 131.83, 129.27, 128.28, 128.14, 128.05, 127.16,

124.94, 122.37, 49.85, 41.57, 18.31. ESI-MS m/z: 334.2 [M+H]⁺; 356.3 [M+Na]⁺;

332.2 [M-H]^-. HRMS calcd for C₂₀H₁₉N₃O₂, [M+H]⁺, 334.14773; found 334.15528.

4.8.3 (R)-N-(3-methyl-1-oxo-1-((pyridin-4-ylmethyl)amino)butan-2-yl)-2-

naphthamide (8c)

1

Light white solid; yield: 71.5%; mp: 106.9–112.8℃. H NMR (400 MHz,

DMSO-d₆) δ 8.73 (t, J = 5.9 Hz, 1H), 8.62 – 8.39 (m, 4H), 8.08 – 7.94 (m, 4H), 7.70 –

7.51 (m, 2H), 7.30 (d, J = 5.9 Hz, 2H), 4.54 – 4.21 (m, 3H), 2.23 (dq, J = 13.4, 6.7 Hz,

1H), 0.99 (dd, J = 15.9, 6.7 Hz, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 173.15,

166.73, 148.97, 148.39, 135.46, 135.23, 134.63, 132.52, 131.84, 129.27, 128.27,

128.13, 128.05, 127.15, 124.93, 123.82, 49.78, 40.26, 18.34. ESI-MS m/z: 362.2

[M+H]⁺; 384.3 [M+Na]⁺; 360.2 [M-H]^-. HRMS calcd for C₂₂H₂₃N₃O₂, [M+H]⁺,

362.17903; found 362.18694.

4.8.4 (R)-N-(2-oxo-1-phenyl-2-((pyridin-4-ylmethyl)amino)ethyl)-2-naphthamide (8d)

1

Light white solid; yield: 65.3%; mp: 123.1–128.4℃. H NMR (400 MHz,

DMSO-d₆) δ 9.09 (d, J = 7.5 Hz, 1H), 8.98 (t, J = 5.9 Hz, 1H), 8.61 (s, 1H), 8.45 (d, J

= 5.7 Hz, 2H), 8.01 (ddd, J = 16.4, 9.7, 4.5 Hz, 4H), 7.67 – 7.53 (m, 4H), 7.38 (dt, J =

23.4, 7.1 Hz, 3H), 7.19 (d, J = 5.5 Hz, 2H), 5.81 (d, J = 7.5 Hz, 1H), 4.37 (d, J = 5.9

13

Hz, 2H). C NMR (101 MHz, DMSO-d₆) δ 170.82, 166.84, 149.85, 148.73, 138.64,

134.69, 132.51, 131.59, 129.34, 128.80, 128.53, 128.31, 128.24, 128.15, 128.04,

127.12, 125.06, 122.39, 58.00, 41.70. ESI-MS m/z: 396.2 [M+H]⁺; 418.3 [M+Na]⁺;

394.2 [M-H]^-. HRMS calcd for C₂₅H₂₁N₃O₂, [M+H]⁺, 396.16338; found 396.17076.

4.8.5 (R)-N-(2-oxo-2-((pyridin-3-ylmethyl)amino)ethyl)-2-naphthamide (8e)

Light white solid; yield: 67.9%; mp: 113.9–121.6℃. ¹H NMR (400 MHz, DMSO-d₆)

δ 9.00 (t, J = 5.8 Hz, 1H), 8.71 – 8.36 (m, 4H), 8.13 – 7.85 (m, 4H), 7.77 – 7.48 (m,

3H), 7.36 (dd, J = 7.7, 4.8 Hz, 1H), 4.36 (d, J = 5.9 Hz, 2H), 3.99 (d, J = 5.9 Hz, 2H).

ESI-MS m/z: 320.0 [M+H]⁺; 342.1 [M+Na]⁺; 318.0 [M-H]^-. HRMS calcd for

C₁₉H₁₇N₃O₂, [M+H]⁺, 320.13208; found 320.13956.

4.8.6 (R)-N-(1-oxo-1-((pyridin-3-ylmethyl)amino)propan-2-yl)-2-naphthamide (8f)

Light white solid; yield: 71.3%; mp: 116.4–124.2℃. ¹H NMR (400 MHz, DMSO-d₆)

δ 8.75 (d, J = 7.1 Hz, 1H), 8.65 – 8.33 (m, 4H), 8.07 – 7.98 (m, 4H), 7.76 – 7.53 (m,

3H), 7.35 (dd, J = 7.4, 4.9 Hz, 1H), 4.58 (p, J = 7.0 Hz, 1H), 4.43 – 4.12 (m, 2H), 1.43

(d, J = 7.2 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 173.15, 166.73, 148.97, 148.39,

135.46, 135.23, 134.63, 132.52, 131.84, 129.27, 128.27, 128.13, 128.05, 127.15,

124.93, 123.82, 49.78, 40.26, 18.34. ESI-MS m/z: 334.2 [M+H]⁺; 356.3 [M+Na]⁺;

332.2 [M-H]^-. HRMS calcd for C₂₀H₁₉N₃O₂, [M+H]⁺, 334.14773; found 334.15515.

4.8.7 (R)-N-(3-methyl-1-oxo-1-((pyridin-3-ylmethyl)amino)butan-2-yl)-2-

naphthamide (8g)

Light white solid; yield: 71.3%; mp: 113.4–121.2℃. ¹H NMR (400 MHz, DMSO-d₆)

δ 8.70 (t, J = 5.8 Hz, 1H), 8.51 (t, J = 7.0 Hz, 3H), 8.42 (d, J = 3.3 Hz, 1H), 8.06 –

8.01 (m, 1H), 7.99 – 7.95 (m, 3H), 7.67 (d, J = 7.9 Hz, 1H), 7.61 – 7.57 (m, 2H), 7.33

(dd, J = 7.7, 4.8 Hz, 1H), 4.34 (t, J = 6.8 Hz, 3H), 2.16 (dd, J = 14.4, 6.8 Hz, 1H),

0.92 (dd, J = 21.5, 6.7 Hz, 6H). ESI-MS m/z: 362.2 [M+H]⁺; 384.3 [M+Na]⁺; 360.2

[M-H]^-. HRMS calcd for C₂₂H₂₃N₃O₂, [M+H]⁺, 362.17903; found 362.18696.

4.8.8 (R)- N-(2-oxo-1-phenyl-2-((pyridin-3-ylmethyl)amino)ethyl)-2-

naphthamide(8h)

1

Light white solid; yield: 68.7%; mp: 124.2–129.1℃. H NMR (400 MHz,

DMSO-d₆) δ 9.07 (d, J = 7.6 Hz, 1H), 8.92 (s, 1H), 8.63 (s, 1H), 8.47 (d, J = 4.5 Hz,

2H), 8.08 (d, J = 6.8 Hz, 1H), 8.02 (d, J = 7.7 Hz, 3H), 7.67 – 7.57 (m, 5H), 7.39 (ddd,

13

J = 12.9, 10.8, 5.9 Hz, 4H), 5.81 (d, J = 7.6 Hz, 1H), 4.40 (s, 2H). C NMR (101

MHz, DMSO-d₆) δ 175.35, 171.38, 159.00, 152.53, 143.41, 141.76, 140.12, 140.03,

139.86, 139.38, 137.24, 136.28, 133.54, 133.01, 131.88, 129.80, 128.66, 128.59,

112.50, 62.59, 45.55. ESI-MS m/z: 396.2 [M+H]⁺; 418.3 [M+Na]⁺; 394.2 [M-H]^-.

HRMS calcd for C₂₅H₂₁N₃O₂, [M+H]⁺, 396.16338; found 396.17026.

4.8.9 (R)-N-(2-oxo-2-((pyridin-4-ylmethyl)amino)ethyl)quinoline-2-carboxamide

(11a)

Light white solid; yield: 69.5%; mp: 102.4–112.7℃. ¹H NMR (400 MHz, DMSO-d₆)

δ 9.19 (t, J = 5.9 Hz, 1H), 8.64 – 8.46 (m, 4H), 8.29 – 8.09 (m, 3H), 7.90 (dd, J = 11.2,

4.1 Hz, 1H), 7.74 (t, J = 7.0 Hz, 1H), 7.30 (d, J = 5.8 Hz, 2H), 4.35 (d, J = 6.0 Hz,

2H), 4.10 (d, J = 6.0 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 169.51, 164.80,

150.21, 149.99, 149.86, 149.75, 148.86, 146.43, 138.26, 131.05, 129.59, 129.33,

128.56, 123.15, 122.51, 121.86, 119.10, 43.03, 41.61. ESI-MS m/z: 321.0 [M+H]⁺;

343.1 [M+Na]⁺; 319.0 [M-H]^-. HRMS calcd for C₁₈H₁₆N₄O₂, [M+H]⁺, 321.12733;

found 321.13374.

4.8.10 (R)-N-(1-oxo-1-((pyridin-4-ylmethyl)amino)propan-2-yl)quinoline-2-

Carboxamide (11b)

Light white solid; yield: 68.2%; mp: 103.9–121.5℃. ¹H NMR (400 MHz, DMSO-d₆)

δ 8.95 (d, J = 7.6 Hz, 1H), 8.79 (t, J = 5.8 Hz, 1H), 8.60 (d, J = 8.5 Hz, 1H), 8.51 (d, J

= 5.1 Hz, 2H), 8.19 (t, J = 8.9 Hz, 2H), 8.10 (d, J = 7.8 Hz, 1H), 7.95 – 7.81 (m, 1H),

7.74 (dd, J = 11.0, 4.0 Hz, 1H), 7.29 (d, J = 5.5 Hz, 2H), 4.67 (p, J = 7.1 Hz, 1H),

4.38 (d, J = 5.9 Hz, 2H), 1.51 (d, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ

172.70, 163.81, 150.04, 148.74, 146.38, 138.44, 132.20, 131.07, 129.65, 129.35,

128.55, 122.43, 118.99, 49.11, 41.64, 19.23. ESI-MS m/z: 335.1 [M+H]⁺; 357.2

[M+Na]⁺; 333.1 [M-H]^-. HRMS calcd for C₁₉H₁₈N₄O₂, [M+H]⁺, 335.14298; found

335.15004.

4.8.11(R)-N-(3-methyl-1-oxo-1-((pyridin-4-ylmethyl)amino)butan-2-yl)quinoline-2-

carboxamide (11c)

Light white solid; yield: 65.1%; mp: 116.5–128.2℃. ¹H NMR (400 MHz, DMSO-d₆)

δ 8.96 (t, J = 5.8 Hz, 1H), 8.73 (d, J = 9.0 Hz, 1H), 8.61 (d, J = 8.4 Hz, 1H), 8.49 (dd,

J = 4.4, 1.6 Hz, 2H), 8.18 (t, J = 7.9 Hz, 2H), 8.11 (d, J = 7.8 Hz, 1H), 7.89 (t, J = 7.0

Hz, 1H), 7.74 (t, J = 7.0 Hz, 1H), 7.27 (d, J = 5.9 Hz, 2H), 4.53 (dd, J = 9.1, 6.4 Hz,

1H), 4.36 (t, J = 6.2 Hz, 2H), 2.22 (dd, J = 13.5, 6.8 Hz, 1H), 0.96 (t, J = 6.5 Hz, 6H).

¹³C NMR (101 MHz, DMSO-d₆) δ 171.33, 163.89, 149.97, 149.82, 148.63, 146.30,

138.65, 131.15, 129.74, 129.41, 128.72, 128.56, 122.63, 118.99, 58.26, 41.64, 31.78,

19.83, 18.52. ESI-MS m/z: 363.2 [M+H]⁺; 385.3 [M+Na]⁺; 361.2 [M-H]^-. HRMS

calcd for C₂₁H₂₂N₄O₂, [M+H]⁺, 363.17428; found 363.18128.

4.8.12 (R)-N-(2-oxo-1-phenyl-2-((pyridin-4-ylmethyl)amino)ethyl)quinoline-2-

carboxamide (11d)

Light white solid; yield: 66.9%; mp: 121.4–121.5℃. ¹H NMR (400 MHz, DMSO-d₆)

δ 9.30 (d, J = 7.7 Hz, 1H), 9.20 (t, J = 5.9 Hz, 1H), 8.64 (d, J = 8.3 Hz, 1H), 8.48 (d, J

= 5.7 Hz, 2H), 8.21 (t, J = 7.4 Hz, 2H), 8.14 (d, J = 8.0 Hz, 1H), 7.93 (t, J = 7.7 Hz,

1H), 7.78 (t, J = 7.5 Hz, 1H), 7.60 (d, J = 7.4 Hz, 2H), 7.47 – 7.38 (m, 3H), 7.19 (d, J

= 5.5 Hz, 2H), 5.81 (d, J = 7.7 Hz, 1H), 4.41 (d, J = 5.8 Hz, 2H). ¹³C NMR (101 MHz,

DMSO-d₆) δ 170.24, 163.34, 149.91, 149.62, 148.32, 146.36, 139.09, 138.71, 131.22,

129.74, 129.48, 129.11, 128.80, 128.57, 128.45, 127.39, 122.39, 118.86, 56.75, 41.75.

ESI-MS m/z: 321.0 [M+H]⁺; 343.1 [M+Na]⁺; 319.0 [M-H]^-. HRMS calcd for

C₂₄H₂₀N₄O₂, [M+H]⁺, 397.15863; found 397.16516.

4.8.13 (R)-N-(2-oxo-2-((pyridin-3-ylmethyl)amino)ethyl)quinoline-2-

carboxamide (11e)

Light white solid; yield: 64.6%; mp: 117.2–124.8℃. ¹H NMR (400 MHz, DMSO-d₆)

δ 9.60 (t, J = 6.2 Hz, 1H), 9.17 (s, 1H), 8.59 (dd, J = 16.0, 5.0 Hz, 2H), 8.47 (dd, J =

4.7, 1.5 Hz, 1H), 8.16 (dd, J = 12.1, 8.6 Hz, 2H), 8.09 (d, J = 7.7 Hz, 1H), 7.92 – 7.84

(m, 1H), 7.79 (d, J = 7.8 Hz, 1H), 7.76 – 7.67 (m, 1H), 7.36 (dd, J = 7.8, 4.8 Hz, 1H),

4.60 (d, J = 6.4 Hz, 2H), 4.07 (d, J = 6.3 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ

168.74, 164.78, 150.46, 149.47, 148.57, 146.48, 138.34, 135.77, 135.46, 131.00,

129.60, 129.30, 128.57, 123.94, 119.18, 41.43, 40.76. ESI-MS m/z: 321.0 [M+H]⁺;

343.1 [M+Na]⁺; 319.0 [M-H]^-. HRMS calcd for C₁₈H₁₆N₄O₂, [M+H]⁺, 321.12733;

found 321.13374.

4.8.14 (R)- N-(1-oxo-1-((pyridin-3-ylmethyl)amino)propan-2-yl)quinoline-2-

carboxamide (11f)

Light white solid; yield: 68.7%; mp: 116.9–128.5℃.¹H NMR (400 MHz, DMSO-d₆)

δ 8.90 (d, J = 7.4 Hz, 1H), 8.73 (s, 1H), 8.57 (d, J = 8.4 Hz, 1H), 8.47 (d, J = 20.3 Hz,

2H), 8.15 (d, J = 8.3 Hz, 2H), 8.08 (d, J = 8.0 Hz, 1H), 7.86 (t, J = 7.3 Hz, 1H), 7.74 –

7.63 (m, 2H), 7.34 (d, J = 5.0 Hz, 1H), 4.74 – 4.50 (m, 1H), 4.35 (d, J = 5.2 Hz, 2H),

13

1.44 (d, J = 6.9 Hz, 3H). C NMR (101 MHz, DMSO-d₆) δ 172.50, 163.73, 150.03,

149.07, 148.55, 146.38, 138.45, 135.36, 135.19, 131.08, 129.66, 129.35, 128.64,

128.55, 123.90, 118.98, 49.02, 40.34, 19.26. ESI-MS m/z: 335.1 [M+H]⁺; 357.2

[M+Na]⁺; 333.1 [M-H]^-. HRMS calcd for C₁₉H₁₈N₄O₂, [M+H]⁺, 335.14298; found

335.15040.

4.8.15 (R)-N-(3-methyl-1-oxo-1-((pyridin-3-ylmethyl)amino)butan-2-yl)quinoline-2-

carboxamide (11g)

Light white solid; yield: 69.4%; mp: 126.4–134.8℃. ¹H NMR (400 MHz, DMSO-d₆)

δ 8.87 (t, J = 5.7 Hz, 1H), 8.70 (d, J = 9.2 Hz, 1H), 8.58 (d, J = 8.6 Hz, 1H), 8.49 (s,

1H), 8.43 (d, J = 3.4 Hz, 1H), 8.16 (dd, J = 8.5, 2.2 Hz, 2H), 8.08 (d, J = 8.1 Hz, 1H),

7.91 – 7.82 (m, 1H), 7.77 – 7.64 (m, 2H), 7.33 (dd, J = 7.7, 4.8 Hz, 1H), 4.48 (dd, J =

9.2, 6.6 Hz, 1H), 4.34 (qd, J = 15.1, 5.8 Hz, 2H), 2.15 (dd, J = 13.5, 6.8 Hz, 1H), 0.90

13

(dd, J = 6.7, 3.5 Hz, 6H). C NMR (101 MHz, DMSO-d₆) δ 171.10, 163.84, 149.83,

149.28, 148.63, 146.33, 138.63, 135.61, 135.11, 131.14, 129.76, 129.42, 128.71,

128.55, 123.90, 118.98, 58.22, 31.84, 19.73, 18.54. ESI-MS m/z: 363.2 [M+H]⁺;

385.3 [M+Na]⁺; 361.2 [M-H]^-. HRMS calcd for C₂₁H₂₂N₄O₂, [M+H]⁺, 363.17428;

found 363.18128.

4.8.16 (R)-N-(2-oxo-1-phenyl-2-((pyridin-3-ylmethyl)amino)ethyl)quinoline-2-

carboxamide (11h)

Light white solid; yield: 70.5%; mp: 128.7–135.2℃. ¹H NMR (400 MHz, DMSO-d₆)

δ 9.27 (d, J = 7.7 Hz, 1H), 9.17 (t, J = 5.9 Hz, 1H), 8.60 (d, J = 8.5 Hz, 1H), 8.44 (dd,

J = 4.5, 1.5 Hz, 2H), 8.17 (dd, J = 8.4, 4.4 Hz, 2H), 8.09 (d, J = 8.0 Hz, 1H), 7.88 (t, J

= 7.1 Hz, 1H), 7.73 (t, J = 7.0 Hz, 1H), 7.56 (d, J = 7.2 Hz, 2H), 7.44 – 7.34 (m, 3H),

7.15 (d, J = 5.8 Hz, 2H), 5.79 (d, J = 7.7 Hz, 1H), 4.38 (d, J = 5.8 Hz, 2H). ¹³C NMR

(101 MHz, DMSO-d₆) δ 170.25, 163.35, 149.95, 149.65, 148.32, 146.37, 139.12,

131.19, 129.74, 129.48, 129.11, 128.89, 128.81, 128.78, 128.56, 127.41, 122.39,

118.86, 56.79, 41.77. ESI-MS m/z: ESI-MS m/z: 397.2 [M+H]⁺; 419.3 [M+Na]⁺;

395.2 [M-H]^-. HRMS calcd for C₂₄H₂₀N₄O₂, [M+H]⁺, 397.15863; found 397.16514.

4.9 In vitro Antifungal activity test

Target compounds were tested for antifungal and antifungal resistance activity,

respectively. The in vitro minimum inhibitory concentrations (MIC) were determined

using the standard guidelines described in the National Committee for Clinical

Laboratory Standards (NCCLS). The MIC value is defined as the lowest concentration

of antifungal inhibitor with inhibitory effect. In the experiment, FLC and Terbinafine

were selected as positive control drugs; all of compounds were dissolved in DMSO and

serially diluted into the growth medium. And the daily growth of fungi was observed

under 35 ℃culture conditions.

4.10 The analysis experiment of the components of C. albicans cells

In this process, the Candida albicans (ATCC SC5314) was selected as the test strain.

FLC and Terbinafine were purchased as the positive control drug. In the experiment,

1640 culture medium was used as the nutrient solution, and the concentration of drug

was set to 16 μg/ml. The wet fungi (0.5 g) treated with different compounds were

weighed, and washed and centrifuged with PBS, and saponified at 80 °C for 90 min.

Then, the saponification solution was extracted with petroleum ether, and washes with

purified water. The solvent was removed under reduced pressure. Finally, the product

was dissolved in methanol solution (10ml), filtered and detected by HPLC.

4.11 Determination of fluorescence spectrum

First, the 2 ml heme solution (40nm/ml) was added in 5 ml quartz pool. Then, the

aqueous solution of target compound 16b was successively titrated, and the mixture

was stirred well. The emission spectrum of 530-575 nm was recorded using 500 nm

as excitation wavelength, the fluorescence intensity used in this paper is considered as

the value after dilution effect.

4.12 ADME/T prediction

The ADME/T properties of compounds 11a and 11b were predicted using the DS

ADMET program in Discovery Studio 3.5. The specific operation process was as

follows: select the “ADMET descriptors” module, and open the parameter browser.

The small molecule compound files were imported. In the parameter settings, the

aqueous solubility, blood brain barrier penetration, CYP2D6 binding, hepatotoxicity,

intestinal absorption and plasma protein binding were chosen as research objects,

respectively.

Acknowledgements

This work was supported by the National Natural Science Foundation of China

(Grant No. 81703357, Grant No. 21473085, Grant No.31701827), the Shandong

Provincial Natural Science Foundation (Grant No. ZR2017BH102) and the Open

Project of Shandong Cllaborative Innovation Center for Antibody Drugs (Grant No.

CIC-AD1837). We also acknowledge Shandong Collaborative Innovation Center for

Antibody Drugs and Engineering Research Center for Nanomedicine and Drug

Delivery systems for this support.

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Highlights



16 new compounds with amide-pyridine scaffolds were designed and synthesized as novel

dual-target antifungal inhibitors.

Compounds 11a and 11b showed better antifungal activity than Fluconazole and Terbinafine.



Compounds 11b exhibits excellent inhibitory activity against drug-resistant pathogenic fungi.

Preliminary mechanism studies of compound 11b were proved.

The target compounds 11a and 11b show excellent drug-forming properties

Graphical abstract: Design and synthesis of amide pyridine derivatives

as novel dual-target (SE, CYP51) antifungal inhibitors.

Article Doi

Design, synthesis and biological evaluation of amide-pyridine derivatives as novel dual-target (SE, CYP51)antifungal inhibitors

DOI: 10.1016/j.bmc.2019.02.009

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.bmc.2019.02.009

Full text of DOI:10.1016/j.bmc.2019.02.009

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