Design, Synthesis and in Vitro Tumor Cytotoxicity Evaluation of 3,5-Diamino-N-substituted Benzamide Derivatives as Novel GSK-3β Small Molecule Inhibitors

Article abstract of DOI:10.1002/cbdv.201900304

Glycogen synthase kinase-3 (GSK-3) plays an important regulatory role in various signaling pathways; such as PI3 K/AKT, which is closely related to the occurrence and development of tumors. At present, the most reported active GSK-3 inhibitors have the same structure: lactam ring or amide structure. To find out the GSK-3β small molecule inhibitor with novel, safe, efficient and more uncomplicated synthesis method, we analyzed in-depth reported crystal-binding patterns of GSK-3β small molecule inhibitor with GSK-3β protein, and designed and synthesized 17 non-reported 3,5-diamino-N-substituted benzamide compounds. Their structures were confirmed by ¹H-NMR, ¹³C-NMR, and HR-MS. The preliminary screening of tumor cytotoxicity of compounds in vitro was detected by MTT, and their structure–activity relationships were illustrated. The results have shown that 3,5-diamino-N-[3-(trifluoromethyl)phenyl]benzamide (4d) exhibited significant tumor cytotoxicity against human colon cancer cells (HCT-116) with IC₅₀ of 8.3 μm and showed commendable selectivity to GSK-3β. In addition, Compound 4d induced apoptosis to some extent and possessed modest PK properties.

Full text of DOI:10.1002/cbdv.201900304

Title: Design, synthesis and in vitro tumor cytotoxicity evaluation of 3,5-
Diamino-N-substituted benzamide derivatives as novel GSK-3β
small molecule inhibitors
Authors: yanping zhou, Lijuan Zhang, Xiujuan Fu, Zhongliang Jiang,
Rongsheng Tong, Jianyou Shi, and Lei Zhong
This manuscript has been accepted after peer review and appears as an
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To be cited as: Chem. Biodiversity 10.1002/cbdv.201900304
Link to VoR: http://dx.doi.org/10.1002/cbdv.201900304

10.1002/cbdv.201900304
Chemistry & Biodiversity
Chem. Biodiversity
Design, synthesis and in vitro tumor cytotoxicity evaluation of 3,5-Diamino-N-
substituted benzamide derivatives as novel GSK-3β small molecule inhibitors
Yanping zhoua, Lijuan Zhanga, Xiujuan Fub, Zhongliang Jiangc, Rongsheng Tonga, Jianyou Shia**, Lei Zhonga*
Yanping Zhou, Lijuan Zhang, Xiujuan Fu contributed equally to this study.
aPersonalized Drug Therapy Key Laboratory of Sichuan Province, Sichuan Academy of Medical Science & Sichuan Provincial People's Hospital, School
of Medicine of University of Electronic Science and Technology of China, No.32 West Second Section First Ring Road, Chengdu, Sichuan, China;
bSchool of pharmacy, Southwest Medicinal University, No.319 Section 3, Zhongshan Road, Luzhou, China;
cDepartment of Hematology, Miller School of Medicine, University of Miami, Miami, USA.
* Corresponding author: Lei Zhong, E-mail: zhl7235301@163.com
**Corresponding author: Jianyou Shi, E-mail: shijianyoude@126.com
Abstract :
Glycogen synthase kinase-3 (GSK-3) plays an important regulatory role in various signaling pathways; such as PI3K/AKT, which is closely related to the
occurrence and development of tumors. At present, the most reported active GSK-3 inhibitors have the same structure: lactam ring or amide
structure. To find out the GSK-3β small molecule inhibitor with novel, safe, efficient and more uncomplicated synthesis method, we analyzed in-depth
reported crystal-binding patterns of GSK-3β small molecule inhibitor with GSK-3β protein, and designed and synthesized 17 non-reported 3,5-diamino-
1
N-substituted benzamide compounds. Their structures were confirmed by H-NMR, 13C-NMR, and HRMS. The preliminary screening of tumor cytotoxic
of compounds in vitro was detected by MTT, and their structure-activity relationships were illustrated. The results have shown that compound 4d
exhibited significant tumor cytotoxic against human colon cancer cells (HCT-116) with IC50 of 8.3 μM and showed commendable selectivity to GSK-3β.
In addition, Compound 4d induce apoptosis to some extent and possess modest PK properties.
Keywords: GSK-3β • small molecule inhibitors • 3,5-diamino-N-substituted benzamide • cytotoxic • structure-activity relationship;
Introduction
Glycogen synthase kinase 3 (GSK-3) is a multifunctional serine/threonine (Ser/Thr) protein kinase composed of two subtypes in mammalian eukaryotic
cells, GSK-3α and GSK-3β, with molecular masses of 51KD and 47KD, respectively. GSK-3α has a longer glycine -rich chain at the N-terminal compare to
GSK-3β.[1, 2] The two subtypes share 36% homology at both ends of the protein and share almost the same sequence in their kinases domain. Besides, it
shares 98% homology at the catalytic sites.[3] GSK-3α is less studied because of its rich glycine amino residues and relatively stable structure. The active
site of GSK-3β consists of two domains: the N-terminal of β-fold and the C-terminal of α-helix, and the ATP binding bag lies between the two
domains.[4, 5] Asp133, Val135 are the primary amino acids in the ATP catalytic site while T ring region and positive regulator Tyr 216 are the main
components in the active site.[6] It consists of the pocket structure of positively charged Arg96, Arg180, and Lys205, but lacks phosphorylated Ser/Thr
residues, which require the substrate to provide pre-phosphorylated Ser/Thr residues.[7, 8] The specific interaction mode between GSK-3β and
substrate can be determined by allowing the active site of GSK-3β to interact with Ser/Thr on the substrate.[9] The biological function of GSK3β is
complex. GSK-3β is involved in insulin signal regulation through phosphorylation of protein kinase B (PKB). GSK-3β is inhibited, and glycogen synthase
(GS) is dephosphorylated and activated to increase the rate of glycogen synthesis[10]. Therefore, the GSK-3 inhibitor can be used as an insulin sensitizer
to inhibit liver glucose release and increase insulin transport. Bussiere et al. reported that GSK-3 β inhibitor compounds 1 and 2 (CHIR611 or CT98014)
enhanced insulin-stimulated glucose transport and improved glucose treatment without increasing insulin levels.[11] In addition, the specific inhibitor
of GSK-3β could mimic the therapeutic effect of emotion stabilizer, which may guide the design of biphasic affective disorder and some
neurodegenerative diseases. Alan P. et al. reported a series of effective and relatively selective GSK-3 inhibitors, 3-benzofuranyl-4-indolylmaleimides,
and proved the effectiveness of the GSK-3β inhibitor in this novel mouse manic model.[12] Also, GSK-3 plays a regulatory role in gene transcription,
accelerating cell cycle, invasion, metastasis and apoptosis of cancer cells.[13, 14] The hypothesis that major upstream kinases associated with GSK-3β are
involved in tumorigenesis has been accepted by the scientific community.[15] Related studies have shown that GSK-3β is the primary regulator of
WinT/Wingless,[16, 17] Hedgehog, PI3K/Akt, and other signaling pathways.[18, 19] As a signal transduction pathway of PI3K/Akt,[20] GSK-3β is used as the
substrate to interact directly with Akt, resulting in the inactivation of GSK-3β phosphorylation.[21, 22] The decrease of GSK-3β-LKB1- Axis protein complex
increase β-catenin accumulation, the acceleration of cell transcription, the up-regulation of c-Myc, c-Jun, Cyclin D1 expression, and the promotion of
1
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tumor cell proliferation.[23] Besides, GSK-3β activates the Wnt signaling pathway through phosphorylated substrates, and the abnormal activation of
Wnt/β-catenin pathway is closely related to the development of colorectal cancer, gastric cancer, and other tumors.[24, 25] GSK-3β promotes the
activation of NF-κB by acting on TNF-α (tumor necrosis factor),[26] promotes the proliferation and differentiation of tumor cells, and enhances the
ability of invasion and metastasis of tumor cells. Therefore, the development of small molecular inhibitors of GSK-3β is one of the hot- spots in the
field of anticancer drugs study.[27, 28] Fass [29]et al. reported some small molecule inhibitors of benzoamide derivatives targeting GSK-3 has the general
formula (structure Ⅰ) and one of the preferred benzalamine derivative (Compound a), as shown in Fig. 1.
Fig.1. Structure of a benzamide derivative with GSK-3β inhibitory activity.
Also, John[30] et al. reported a series of 2-amino-substituted derivatives as potential inhibitors of IKK-α and IKK-β kinases (Fig.2). In addition to the
selectivity of IKK-α and IKK-β kinases, these compounds also have better overall kinase selectivity. Among them, compound b showed excellent
selectivity to GSK-3β, CDK2, ALK5, EGFR, ErbB2, PLK1, VEGFR2, etc..[31] Interestingly, the kinase docking result of compound c with CDK2 showed that
the carbonyl oxygen atoms and amino groups on the amines, as well as the amino groups in the parent structure of the compound c, can form
hydrogen bonds with the active sites Cys 99 and Glu 97 of CDK2, which indicated that the amide and amino group on the phenyl ring were the
important groups to inhibit the CDK2 kinase activity.[32] By analyzing the structure of the currently reported well-active GSK-3β small molecule
inhibitors, such as 1-Azakenpaulllone (IC50=0.018μM),[33] AR-A014418 (IC50=0.104 µM),[34, 35] Compound 21 (IC50=0.007 μM),[36] Compound 11 (IC50=0.126
μM),[37] hymenialdisine (IC50=0.035 μM),[38, 39] Compound 9i (IC50=0.019 μM)[7] (Fig. 3), we found GSK-3β inhibitors mainly structured in an amide or
lactam. Related studies have also shown that some compounds use two water molecules as intermediate bridges, linking Gln185 and Thr138 to form
hydrogen bonds.[40] Therefore, we hypothesized that the ability of amino acid residues in the catalytic domain of GSK-3β forming hydrogen bonds
with the amide or lactam structures on the candidate molecules should be the focus of GSK-3β small molecule inhibitor studies.
Fig.2. Structure of compounds b and c.
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Fig.3. Part reported GSK-3β small molecule inhibitors.
To obtain a novel small molecule inhibitor of GSK-3β, we performed a virtual computer screening in a library of 3 million compounds according to the
reported crystal structure of GSK-3β protein and selected 10% of the molecules with top scores. In combination with our hypothesis, 17 unreported 3,5-
1
diamino-N-benzamides (compounds 4a-q) were synthesized. Structural characterization was performed by H NMR, 13C-NMR, and HRMS. The tumor
cytotoxic was measured in vitro by MTT assay, and its selectivity was determined by kinase assay. Meanwhile, Flow Cytometry was used to detect the
apoptosis cell distribution after compounds exposures, and the tested compounds’ pharmacokinetic parameters were determined by HPLC-MS. Our
study lays a foundation for the research of novel GSK-3β small molecule inhibitors.
Results and Discussion
1. Chemistry
The 3,5-diamino-N-substituted-benzamide compounds were synthesized as the synthetic route. As shown in Scheme 1, the 3,5-dinitrobenzoic acid
(compound 1) and series of amines with different substituents as a starting material, then via the nucleophilic attack of amines with acid chlorides to
obtain a series of 3,5-dinitro-N-substituted benzamide compounds (compound 3), which were reduced by hydrazine hydrate to get 3,5-diamino-N-
substituted benzamide compounds (compounds 4a-q). All the objective compounds were confirmed by 1H-NMR, 13C-NMR, and HRMS.
Compound
R
Compound
R
Compound
R
Compound
R
4a
4b
4c
4e
4f
4i
4m
4n
4o
4p
4j
4k
4l
4g
4h
4d
4q
Scheme 1. Synthesis of 3,5-diamino-N-substituted-benzamide compounds 4a-q. Reactions and conditions: (a) DMF, SOCl2, 90°C, reflux; (b) amines with
different substituent, THF, DIEA, 0°C -RT; (c)N2H4•H2O, ethanol, Pd/C, 90°C, reflux.
3
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2. Tumor cytotoxic of compounds 4a-q
MTT assay was applied to measure the tumor cytotoxic of target compounds 4a-q in vitro against three tumor cell lines, including human metastatic
pancreatic cancer cell lines (AsPC-1), human colorectal cancer cell lines (HCT-116), and human glioma cell line (U251).[41] Doxorubicin was used as the
positive control. As shown in Table 1, the compounds 4a-q exhibited different in vitro anti-proliferative effects on the three tumor cell lines as the
substituents vary. Among them, compound 4d showed the highest inhibitory effects on all three tumor cell liness, with the IC50 of 12.5, 8.3, and 11.2
μM for AsPC-1, HCT-116, and U251, respectively. Compounds 4c, 4e, and 4f were next. Compounds 4a, 4b, 4g, 4i, 4k, 4o, and 4p exhibited some tumor
cytotoxicity against a particular cell line, while compounds 4h, 4j, 4l, 4m, 4n, 4q showed no significant tumor cytotoxicity. Among these comp0unds,
it is worth noting that only compound 4p (IC50 = 15.35 µM) has certain cytotoxicity to normal human renal epithelial cells (293T), with significant
selectivity (IC50 ˃ 100µM). Together, these results showed the orientation of drug design. Compound 4d will conduct further research.
Table1. Tumor cytotoxic assay data for compounds 4a-q
compound
IC50 (μM)a
AsPC-1
NA
HCT-116
56.9
48.9
36.3
8.3
U251
293T
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
15.35
NA
1.04
4a
NA
4b
59.4
23.5
12.5
35.9
27.1
NA
NA
4c
69.7
11.2
40.5
52.8
NA
4d
4e
35.2
28.1
59.8
NA
4f
4g
4h
NA
NA
4i
78.3
NA
37.7
NA
NA
4j
NA
4k
37.3
NA
NA
51.8
NA
4l
NA
4m
NA
NA
NA
4n
NA
NA
NA
4o
NA
47.9
NA
NA
4p
18.5
NA
35.5
NA
4q
NA
Doxorubicin
0.74
0.53
0.96
IC50: The concentration of the compound at which 50% of the target cells are inhibited. The values are presented as the mean ± SD (standard error of
the mean) from three separated experiments.
NA: no activity, IC50˃100µM
3. Kinase assay
Kinase selectivity is a critical factor in the effectiveness of GSK-3β small molecule inhibitors.[42, 43] Thus, to evaluate the kinase selectivity of compound
4d, six kinases were measured and shown in Table 2. Among them, Aurora-A and PLK1 are two common Ser/Thr kinases that widely exist in eukaryotic
cells. Besides, JAK1 is the key kinase of STAT 3, Akt and GSK-3 β signal transduction. Furthermore, Fumi et al. reported MEK1/2 induces Tyr216
phosphorylation on GSK-3β and triggers GSK-3β nuclear translocation.[44] FLT- 3 is a receptor tyrosine kinase that plays a crucial role in regulating
hematopoietic progenitor cells. The most common tyrosine kinase mutation in acute myeloid leukemia (AML) is the internal tandem duplication (ITD)
mutation of FLT- 3[22]. Inhibiting PI3K/Akt/GSK- 3 pathway downstream of FLT3-ITD down-regulated DNA damage and significantly enhanced apoptosis
induction. Compound 4d displays an evident selectivity among other kinases with IC50 at 5.2 µM for GSK-3β while 385µM, 5820µM for JAK1 and MEK1
respectively. However, we found that Compound 4d also showed particular inhibition on FLT-3 kinase, which may be due to the negative regulation
FLT- 3 kinase by GSK3 inhibitor. However, the specific mechanism needs to be further studied and confirmed. It needs to be further optimized to
obtain a more selective small inhibitor.
Table2. Enzyme assay data for compound 4d IC50 (μM)
Aurora-A
235
PLK1
190
GSK-3β
FLT-3
8.3
JAK1
385
MEK1
5820
4d
5.2
4. Analysis of apoptosis by Flow Cytometry
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Inducing apoptosis is an important strategy for cancer treatment. In this study, Flow Cytometry-based AnnexinV-FITC/Propidium Iiodide (PI) assay was
applied to determine whether compound 4d induced apoptosis in the tested cancer cell line.[45] In the early stage of apoptosis, phosphatidyl serine
(PS), which was located in the medial membrane of the cell membrane, was transferred to the outside of the cell membrane and could bind to the
Annexin V while PI can stain the nuclei of late apoptotic and necrotic cells.[46] The results of Annexin V-PI double staining detected by Flow Cytometry
can be used to analyze cell apoptosis[47]. As shown in Fig. 4, compared with control, 5 µM of compound 4d exposure significantly increased the
percentage of total apoptotic cells (right quadrants, Q2+Q4). The 10 µM of compound 4d exposure increased the percentage of total apoptotic cells
to 16.38%. Increasing the compound 4d dose further to 15 µM, the percentage of apoptotic cells increased to 30.11%. However, the number of late
apoptotic/necrotic cells (Q1) also increased from 1.11% (control) to 13.11% (15 µM). The above results indicate compound 4d exposure results in cell
death might through inducing apoptosis in combination with other pathways. These results also suggest that compound 4d has a weaker influence on
cell apoptosis.
Control
5µM
10µM
15µM
Fig. 4. Flow cytometry-based apoptosis detection in HCT-116 cells after compound 4d exposure (5, 10, 15 µM).
5. Pharmacokinetic study
Compound 4d was tested in rat for its PK profile as is shown in Table 3. The compound exhibited low plasma clearance in rat compared to liver blood
flow. Time of maximal concentration (Tmax) occurred consistently early in animal, indicating relatively fast oral absorption. Compound 4d exhibited
moderate half-life (T1/2) as well as higher plasma clearance of 1.691 L/h/kg, and it also showed a high systemic exposure with areas under the curves
(AUC) 5755.550 µg/L*h. However, the plasma concentration decreased slightly around 4-5 h and with a higher rate in 12-17 h (Fig. 5), which may be
related to the reversible binding of drugs to plasma proteins. Overall, compound 4d possesses modest PK properties.
Table 3. PK parameters of compound 4d
compound dose (mg/kg) AUC0−24h (µg/L*h) Cmax (µg/L) Tmax
t1/2 (h) Vss (L/kg) CL (L/h/kg)
11.009 1.691
4d 10 5755.550 983.400 2.000 4.512
Fig.5. C-T curve of compound 4d.
6. Discussion
The IC50 values in Table 1 indicate the compounds 4a-q showed different degrees of inhibitory effects on the tested three tumor cell lines. These 17
compounds share the same parent core structure, 3,5-dinitro-N-substituted benzamide. Therefore, the structure of the substituent R is a crucial factor
affecting the compound’s in vitro tumor cytotoxicity. By analyzing the compound structure and its inhibitory effects, we found that the bulkier the
substituent R group, the higher tumor cytotoxicity of the compound. Since the compound 4b (R=3-CH3Ph) showed a higher inhibitory effect compare
to the compound 4j (R=CPE), we can conclude that a compound has an aromatic ring should exhibit higher inhibitory effect compare to a compound
has an aliphatic saturated cyclic alkane such as cyclopentane. Besides, when the substituent R is an aromatic benzene ring, the nature of the
substituent and its position on the phenyl ring also affect the tumor cytotoxicity of the compound. Also, when fixing the position of the substituent R,
an electron-withdrawing group has a stronger tumor cytotoxic effect compare to an electron-repelling group. What is more, the stronger electron-
absorbing ability of the substituent R group, the higher tumor cytotoxicity of the compound. For example, the activity of compound 4d (R=CF3Ph) is
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higher than that of compound 4c (R=-CH2Ch3Ph). Compound 4d (R = CF3Ph) is more active than compound 4m (R = F). When the substituents are of
the same species, and the positions are different, the activity of the compound 4b (R=3-CH3Ph) is higher than that of the compound 4i (R=4-CH3Ph).
Compound 4F (R=4-ClPh) has higher tumor cytotoxicity than compound 4P (R=2-ClPh), and an active sequence can be inferred: meta-substituted ˃
para-substituted ˃ortho-substitution. When the substituents are in the same position, and the substituents are all heterocycles, tumor cytotoxicity is
decreased with more heteroatoms on the ring. For example, compound 4g is more effective than compound 4h. The analysis of the overall activity
data indicate that the effect of the substituent on the tumor cytotoxicity of the compound is more likely to be the nature of the substituent rather
than the position of the substituent. Based on the above discussions and analysis of the structure-activity relationship of the target compound, we
can draw the following conclusions.
(1) When the substituent R is an aromatic benzene ring, the effect of the position of the substituent on the phenyl ring against the compound tumor
cytotoxicity is: meta-substituent˃ para-substituent˃ ortho-substituent;
(2) When the substituent R is an aromatic benzene ring, the effect of the substituent on the benzene ring on the compound tumor cytotoxicity is:
electron-withdrawing group ˃electron-donating group;
(3) When the substituent R is an aromatic benzene ring, the type and number of substituents on the benzene ring have a more significant effect on
the compound tumor cytotoxicity than the substituted position;
(4) The bulky substituent R group increases the compound’s tumor cytotoxicity.
Conclusions
1
We designed and synthesized a series of unreported 3,5-diamino-N-benzamides, which were confirmed by H NMR, 13C-NMR, and HRMS. The in vitro
tumor cytotoxicity of these compounds was evaluated using three human tumor cell lines (ASPC-1, HCT-116, U251). The results showed that the
compound 4a-q had different inhibitory activities against all three tumor cell lines. Among them, compound 4d showed significant inhibitory effect on
human colon cancer cell HCT-116 (IC50=8.3μM), which has substantial reference value for the study of novel GSK-3β small molecule inhibitor.
Apoptosis analysis showed that compound 4d induced apoptosis to some extent. In vivo experiments have demonstrated that compound 4d has an
acceptable overall character (selectivity, ADME). In summary, compound 4d has significant guiding value for finding novel GSK-3β small molecule
inhibitors.
Experimental Section
1. General Information
All chemical reagents were purchased from the commercial suppliers (Chengdu Kelong Chemical Reagent Factory, Chengdu, China) and used without
further purification. The reactions were monitored by thin layer chromatography (TLC) carried out on precoated silica plates. The 1H-NMR and 13C-
NMR spectra were performed on a Bruker Avance 400 NMR spectrometer in DMSO-d6 with Tetramethylsilane(TMS) as internal reference, chemical
shifts (δ) are reported in ppm referenced to 1H (DMSO-d6 δ=2.50 ppm), 13C (DMSO-d6 δ=39.52 ppm), the abbreviation for signal were expressed as
follows: s=singlet, d =doublet, dd=doublet of doublets, t=triplet, q=quartet, m=multiplet, and coupling constants are represented in Hz. HRMS spectra
were obtained on a Waters UPLC/Q-TOF-MS instrument. HPLC column was purchased from Diamonsil (Lake Forest, CA 92630 USA). The purity of each
compound 4a-q was determined on Waters e2695 HPLC (column: Diamonsil (Lake Forest, CA 92630 USA) methanol (60%-90%); low rate, 1.0 mL/min;
UV wavelength, 210−400 nM; temperature, 25 °C; volume, 10 μL). 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) was purchased
from BioFroxx (Guangzhou saiguo biotech Co., LTD, Guangdong, China). and Annexin V-FITC/PI apoptosis detection kit was purchased from BD
Biosciences Pharmingen (San Diego, US). Human tumor cell lines were all provided by the State Key Laboratory of Biotherapy, Sichuan University. The
experimental rats were from the Institute of Experimental Animals, Sichuan people's Hospital, Sichuan Academy of Medical Sciences.
2. Synthesis of 3,5-diamino N-substituted benzamide compounds 4a-q
2.1 Synthesis of key intermediates compound 3
3,5-dinitrobenzoic acid (compound 1, 5 mmol, 1.06 g) dissolved in SOCl2 (15 mL) and N, N-dimethylformamide (DMF, 1.5 mL), 90°C, reflux. The progress
of the reaction was monitored using TLC. After the reaction was completed, add appropriate amount of CH2Cl2 to remove SOCl2, tetrahydrofuran
(THF,10 mL) dissolved to give compound 2. Then compound 2 was added slowly into the colded mixture solution at 0°C with aniline (5 mmol, 456 μL),
N, N-diisopropylethylamine (DIEA, 1.5 mL), after 15 minutes, move the reaction to room temperature agitator, the reaction was also recored by TLC.
After reaction completed, evaporated the solvent, extracted with ethyl acetate (15 mL× 4) and washed with saturated salt water, dried over
anhydrous Na2SO4 and concentrated the organic phase to give the compound 3.
2.2. Synthesis of 3,5-diamino N-substituted benzamide compounds 4a-q
Hydrazine hydrate (20 mmol, 1mL), appropriate amount of Pd/C were added to compound 3 dissolved in absolute ethanol, 90°C, reflux. The reaction
was monitored via TLC. When reaction completed, pumping filter, spin the filtrate to give the crude product. The pure compound 4a (compound 4a:
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3,5-diamino-N- phenylbenzamide) was obtained by recrystallization from absolute ethanol-water recrystallization. Compound 4b-q was synthesized in
the same method as compound 4a.
Compound 4a: 3,5-diamino-N-phenylbenzamide Yield: 82%. Melting point: 182.5-184.9°C. Purity: 97.70%. 1H-NMR (400 MHz, DMSO) δ 9.92 (s, 1H), 7.99 –
7.58 (m, 2H), 7.49 – 7.11 (m, 2H), 7.20 – 6.79 (m, 1H), 6.29 (d, J = 2.0 Hz, 2H), 5.99 (t, J = 2.0 Hz, 1H), 4.94 (s, 4H).13C-NMR (101 MHz, DMSO-d6) δ 167.74,
149.58, 140.09, 137.34, 128.93, 123.53, 120.43, 102.76,102.70. HRMS (ESI) m/z: (M+H)+ calcd for C13H13N3O 227.2670, found 228.1102.
Compound 4b: 3,5-diamino-N-(m-tolyl)benzamide Yield: 79%. Melting point: 153.6-155.8°C. Purity: 98.48%. 1H-NMR (400 MHz, DMSO) δ 9.86 (s, 1H),
7.60 (d, J = 1.9 Hz, 1H), 7.51 (dd, J = 8.1, 2.0 Hz, 1H), 7.18 (t, J = 7.8 Hz, 1H), 6.86 (d, J = 7.5 Hz, 1H), 6.32 (d, J = 2.0 Hz, 2H), 6.03 (d, J = 2.1 Hz, 1H), 5.19 (s,
4H), 2.28 (s, 3H).13C-NMR (101 MHz, DMSO-d6) δ 167.50 , 148.82, 139.95, 138.03, 137.44, 128.77, 124.30, 121.00, 117.68, 103.55, 103.43, 21.73. HRMS (ESI)
m/z: (M+H)+ calcd for C14H15N3O 241.2940, found 242.1287.
Compound 4c: 3,5-diamino-N-(3-ethylphenyl)benzamide Yield: 79%. Melting point: 200.5- 201.8°C. Purity: 98.07%. 1H-NMR (400 MHz, DMSO) δ 9.84 (s,
1H), 7.76 – 7.49 (m, 2H), 7.29 – 6.91 (m, 2H), 6.28 (d, J = 2.0 Hz, 2H), 5.98 (t, J = 2.0 Hz, 1H), 4.92 (s, 4H), 2.55 (q, J = 7.6 Hz, 2H), 1.16 (t, J = 7.6 Hz,
3H).13C-NMR (101 MHz, DMSO-d6) δ 167.53, 149.55, 138.92, 137.77, 137.42, 128.12, 120.53, 102.76, 102.65, 28.10, 16.23. HRMS (ESI) m/z: (M+H)+ calcd for
C15H17N3O 255.3210, found 256.1468.
Compound 4d: 3,5-diamino-N-(3-(trifluoromethyl)phenyl)benzamide Yield: 80%. Melting point: 188.4-189.0°C. Purity: 97.06%. 1H-NMR (400 MHz, DMSO)
δ10.28 (s, 1H), 8.23 (d, J = 1.9 Hz, 1H), 8.01 (dd, J = 8.2, 2.0 Hz, 1H), 7.55 (t, J = 8.0 Hz, 1H), 7.39 (d, J = 7.6 Hz, 1H), 6.31 (d, J = 2.0 Hz, 2H), 6.01 (t, J = 2.0 Hz,
1H), 4.98 (s, 4H).13C-NMR (101 MHz, DMSO-d6) δ 168.13, 149.68, 140.87, 136.72, 130.17, 129.89, 129.55, 123.85, 123.32, 119.81, 116.42, 116.38, 102.95, 102.73.
HRMS (ESI) m/z: (M+H)+ calcd for C14H12F3N3O 295.2652, found 296.0992.
Compound 4e: 3,5-diamino-N-(4-fluorophenyl)benzamide Yield: 81%. Melting point: 153.6- 155.1°C. Purity: 98.84%. 1H-NMR (400 MHz, DMSO) δ 10.28 (s,
1H), 8.23 (s, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.55 (t, J = 8.4 Hz, 1H), 7.39 (d, J = 7.6 Hz, 1H), 6.31 (d, J = 1.6 Hz, 2H), 6.01 (s, 1H), 4.98 (s, 4H).13C-NMR (101 MHz,
DMSO-d6) δ 167.60, 159.61, 157.23, 149.38, 137.15, 136.45,136.43, 122.20,122.121,115.58,115,36,102.96. HRMS (ESI-MS) m/z: (M+H)+ calcd for C13H12FN3O
245.2574, found 246.1059.
Compound 4f: 3,5-diamino-N-(4-chlorophenyl)benzamide Yield: 83%. Melting point: 196.5-197.3°C. Purity: 97.09%. 1H-NMR (400 MHz, DMSO) δ 10.08 (s,
1H), 8.14 – 7.61 (m, 2H), 7.53 – 7.12 (m, 2H), 7.53 – 7.12 (m, 2H), 6.00 (t, J = 2.0 Hz, 1H), 4.98 (s, 4H).13C-NMR (101 MHz, DMSO-d6) δ 167.82, 149.56,
139.06, 137.02, 128.85, 127.11, 121.90, 102.87, 102.80. HRMS (ESI) m/z: (M+H)+ calcd for C13H12ClN3O 261.7090, found 262.0741.
Compound 4g: 3,5-diaminophenyl)(piperidin-1-yl)methanone Yield: 74%. Melting point: ＞220°C . Purity: 93.02%. 1H-NMR (400 MHz, DMSO) δ5.82 (t, J =
2.0 Hz, 1H), 5.72 (d, J = 2.0 Hz, 2H), 4.85 (s, 4H), 3.47 (s, 2H), 3.30 (s, 2H), 1.64 – 1.34 (m, 6H). 13C-NMR (101 MHz, DMSO-d6) δ 170.60, 149.56, 138.17,
101.35, 100.44, 48.27, 42.36, 26.61, 25.91, 24.61. HRMS (ESI) m/z: (M+H)+ calcd for C12H17N3O 219.2880, found 220.1438.
Compound 4h: 3,5-diaminophenyl)(morpholino)methanone Yield: 79%. elting point: ＞220°C . Purity: 99.61%. 1H-NMR (400 MHz, DMSO) δ5.84 (t, J =
2.0 Hz, 1H), 5.75 (d, J = 2.0 Hz, 2H), 4.88 (s, 4H), 3.64 – 3.48 (m, 6H), 3.33 (s, 2H).13C-NMR (101 MHz, DMSO-d6) δ 170.96, 149.40, 136.88, 101.81, 100.81,
70.11, 66.75. HRMS (ESI) m/z: (M+H)+ calcd for C11H15N3O2 222.1237, found 222.1235.
Compound 4i: 3,5-diamino-N-(p-tolyl)benzamide Yield: 82%. Melting point: 209.9-210.7°C. Purity: 98.73%. 1H-NMR (400 MHz, DMSO) δ9.83 (s, 1H), 7.81 –
7.45 (m, 2H), 7.10 (d, J = 8.2 Hz, 2H), 6.27 (d, J = 2.1 Hz, 2H), 5.98 (t, J = 2.0 Hz, 1H), 4.92 (s, 4H), 2.26 (s, 3H). 13C-NMR (101 MHz, DMSO-d6) δ 167.53,
149.55, 137.58, 137.43, 132.39, 129.32, 120.43, 102.75, 102.63, 20.94. HRMS (ESI) m/z: (M+H)+ calcd for C14H15N3O 241.2940, found 242.1279.
Compound 4j: 3,5-diamino-N-cyclopropylbenzamide Yield: 73%. Melting point: 208.5-210.0°C. Purity: 85.89%. 1H-NMR (400 MHz, DMSO) δ8.59 (d, J = 4.3
Hz, 1H), 7.81 – 7.65 (m, 1H), 7.48 (t, J = 2.2 Hz, 1H), 7.42 – 7.38 (m, 1H), 5.98 (s, 2H), 2.90 – 2.79 (m, 1H), 1.43 – 1.06 (m, 2H), 0.73 – 0.64 (m, 2H), 0.61 –
0.54 (m, 2H).13C-NMR (101 MHz, DMSO-d6) δ 166.62, 150.35, 149.10, 136.96, 118.91, 109.57, 108.74, 23.62, 6.11. HRMS (ESI) m/z: (M+H)+ calcd for
C10H13N3O 192.1131, found 192.0602.
Compound 4k: 3,5-diamino-N-(3-methoxyphenyl)benzamide Yield: 67%. Melting point: 162.4-163.2°C. Purity: 98.49%. 1H-NMR (400 MHz, DMSO) δ 10.37
(s, 1H), 9.15 – 8.78 (m, 1H), 7.88 (dd, J = 2.1, 1.5 Hz, 1H), 7.56 (t, J = 2.2 Hz, 1H), 7.51 – 7.33 (m, 4H), 7.26 (t, J = 8.1 Hz, 1H), 6.71 (dddd, J = 8.2, 5.6, 2.6, 0.9
Hz, 1H), 6.09 (s, 2H), 3.76 (s, 3H). 13C-NMR (101 MHz, DMSO-d6) δ 164.70, 159.89, 150.67, 149.16, 140.52, 137.33, 129.86, 119.32, 113.14, 109.99, 109.84,
109.05, 106.67, 55.49. HRMS (ESI) m/z: (M+H)+ calcd for C14H15N3O2 258.1237, found 258.1179.
Compound 4l: 3,5-diamino-N-(4-methoxyphenyl)benzamide Yield: 75%. Melting point: 195.2-196.8°C. Purity: 98.64%. 1H-NMR (400 MHz, DMSO) δ 10.28
(s, 1H), 7.91 – 7.85 (m, 1H), 7.69 – 7.64 (m, 2H), 7.58 – 7.46 (m, 2H), 7.51 – 7.46 (m, 3H), 6.07 (s, 2H), 3.75 (s, 4H).13C-NMR (101 MHz, DMSO-d6) δ 164.24,
156.22, 150.63, 149.13, 137.46, 132.35, 122.88, 119.29, 114.23, 109.83, 108.97, 55.40. HRMS (ESI) m/z: (M+H)+ calcd for C14H15N3O2 258.1237, found
258.1238.
Compound 4m: 3,5-diamino-N-(3-fluorophenyl)benzamide Yield: 72%. Melting point: ＞220°C. Purity: 97.64%. 1H-NMR (400 MHz, DMSO) δ 10.58 (s, 1H),
7.88 ((t, J = 1.8 Hz, 1H), 7.80 – 7.65 (m, 2H), 7.56 (dt, J = 8.1, 1.6 Hz, 2H), 7.50 – 7.46 (m, 1H), 7.45 – 7.35 (m, 2H) , 6.95 (td, J = 8.5, 2.7 Hz, 1H), 6.11 (s, 2H).
13C-NMR (101 MHz, DMSO-d6) δ 164.95, 161.29, 150.72, 149.18,141.16, 141.05, 137.02, 130.78, 130.69, 119.25, 116.57, 110.17, 109.05. HRMS (ESI) m/z: (M+H)+
calcd for C13H12FN3O 246.1037, found 246.1040.
7
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10.1002/cbdv.201900304
Chemistry & Biodiversity
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Compound 4n: 3,5-diamino-N-(2,4-difluorophenyl)benzamide Yield: 77%. Melting point: 201.8-202.5°C. Purity: 97.18%. 1H-NMR (400 MHz, DMSO) δ 10.31
(s, 1H), 7.92 – 7.90 (m, 1H), 7.59 – 7.55 (m, 2H), 7.52 – 7.50 (m, 1H), 7.41 – 7.33 (m, 2H), 7.17 – 7.09 (m, 2H), 6.10 (s, 2H).13C-NMR (101 MHz, DMSO-d6) δ
164.62,153.77, 150.70, 149.15, 136.06, 135.67,119.27,118.20,112.69,110.24,109.38,109.06. HRMS (ESI) m/z: (M+H)+ calcd for C13H11F2N3O 264.0943, found
264.0940.
Compound 4o: 3,5-diamino-N-(3-chloro-4-fluorophenyl)benzamide Yield: 63%. Melting point: ＞220°C. Purity: 86.94%. 1H-NMR (400 MHz, DMSO) δ
10.57 (s, 1H), 8.98 – 8.79 (m, 1H), 8.09 – 8.01 (m, 1H), 7.91 – 7.87 (m, 1H), 7.80 – 7.68 (m, 2H), 7.57 (t, J = 2.2 Hz, 1H), 7.50 – 7.39 (m, 2H), 6.11 (s, 2H).13C-
NMR(101 MHz, DMSO-d6) δ 164.78, 150.73, 149.15, 136.79, 122.39,121.29,121.22, 119.26, 117.42,117.20, 110.23, 108.96. HRMS (ESI) m/z: (M+H)+ calcd for
C13H11ClFN3O 280.0647, found 280.0602.
Compound 4p: 3,5-diamino-N-(2-chlorophenyl)benzamide Yield: 69%. Melting point: 184.3-185.7 °C. Purity: 90.59%. 1H-NMR (400 MHz, DMSO) δ 10.27 (s,
1H), 8.32 – 7.74 (m, 2H), 7.64 – 7.50 (m, 4H), 7.36 (dtd, J = 32.2, 7.6, 1.7 Hz, 3H), 6.10 (s, 2H).13C-NMR (101 MHz, DMSO-d6) δ 164.73, 150.72, 149.22,
136.44, 135.30, 130.23, 129.11, 128.22, 127.99, 119.28, 110.18, 109.07. HRMS (ESI-MS) m/z: (M+H)+ calcd for C13H12ClN3O 262.0741, found 262.0737.
Compound 4q: 3,5-diamino-N-(6-chloropyridin-3-yl)benzamide Yield: 71%. Melting point:＞220°C. Purity: 87.52%. 1H-NMR (400 MHz, DMSO) δ 10.73 (s,
1H), 8.78 (d, J = 2.7 Hz, 2H), 8.24 (dd, J = 8.7, 2.8 Hz, 2H), 7.92 (t, J = 1.8 Hz, 1H), 7.59 (t, J = 2.2 Hz, 1H), 7.56 – 7.45 (m, 2H), 6.13 (s, 2H).13C-NMR (101 MHz,
DMSO-d6) δ 165.05, 150.77, 149.33, 144.72,142.05, 136.30, 135.61, 131.56, 124.55, 119.40, 110.21, 109.05. HRMS (ESI) m/z: (M+H)+ calcd for C12H11ClN4O
263.0694, found 263.0697.
3. Tumor cytotoxic of compounds 4a-q
The compounds 4a-q were evaluated for their cell proliferation inhibitory activity (%) in vitro against human cancer cell lines, including human
metastasis of pancreatic cancer cell lines (AsPC-1), human colorectal cancer cell lines (HCT-116), and human glioma cells (U251) and a normal human
renal epithelial cell (293T). The cells in good condition were seeded in 96-well plates at 4×103 cells/well and incubation overnight. Compound 4a-q was
diluted by gradient dilution to 100, 50, 25, 12.5, 6.25 and 3.125 μM. Then 100μL of diluted drug solution was added to each well and set 3 replicate wells
for each concentration, 100μL of complete medium was added to the control group, meanwhile, and Doxorubicin hydrochloride was used as the
positive control, diluted according to compound 4a-q. Plates were placed under 5% CO2 at 37 °C and cultured for 48 h in the incubator. Then each well
was treated with 20μL MTT solution (5 mg/mL) and continue incubation for 4 h. The supernatant was discarded, and 150μL of DMSO was added to
each well. The absorbance (A) was measured at 570 nm, calculate the inhibition rate of the target compound on tumor cells according to the
following formula.
Relative cell proliferation inhibition rate (%) = (control group A570 - experimental group A570)/control group A570 ×100%. SPSS software was used to
calculate half-value inhibition (IC50).
4. Kinase assay
The kinase assay of compound 4d was measured using Kinase Profiling services provided by Wizards Chemical.
5. Analysis for Apoptosis by Flow Cytometry
Apoptosis was detected using an Apoptosis Detection Kit. Cells were cultured in 6-well plates (4 × 105 cells per well) and incubated at 37°C for 24 h and
then cells were given compound 4d at 5,10 and 15 µM for incubation 24h. After that, the cells were digested with trypsin without EDTA for a while,
collected and centrifuged for 5 minutes at 1000 r/min. Cells were washed twice with precooled PBS and suspended with100 μL 1×Binding Buffer. Mix
gently with 5 μL Annexin V-FITC and 10 μL PI staining solution for 15 min at room temperature in the dark. After adding 400μL 1 × Binding Buffer, the
sample was mixed and placed on ice. The sample was detected by flow cytometry within 1 hour.
6. Pharmacokinetic study
In vivo PK studies were conducted in Sprague Dawley rats (6, 180-220g, half male and half female). No food but with water for 12 h before the
experiment. Compound 4d was dissolved in 5% DMSO, 5% solutol, and 90% saline and administered to rats via gavage at 10 mg/kg.[48] Blood samples
were collected serially at scheduled times (5min, 1h, 1.5h, 2h, 2.5h, 3h, 4h, 5h, 6h, 8h, 10h,12h,16h, 20h, 24h) within 24 hours after dosing and placed in
tubes containing heparin sodium.[49] Plasma samples were centrifuged for 2 min, at 4°C for 12000 rpm. After centrifugation, plasma was taken into the
new tube and stored at -80°C for bioanalysis.[50, 51] Plasma concentration was measured by LC-MS/MS and analyzed by DAS 2.0 atrioventricular model.
Acknowledgements
The financial support of this study was from the Key Project of Clinical Research and Transformation Fund of Sichuan people's hospital (No.2016LZ03)
and National Natural Science Foundation of China (No.81703457). The project of Chengdu science and technology bureau (No.2015-HM01-00463-SF),
Special research project on TCM science and technology in Sichuan province (No.2016Q001) and the National Key Specialty Construction Project of
Clinical Pharmacy (30305030698) is gratefully acknowledged.
Author Contribution Statement
8
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10.1002/cbdv.201900304
Chemistry & Biodiversity
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Yanping Zhou, Lijuan Zhang, Xiujuan Fu participated in the whole process of the experiment. Zhongliang Jiang, Rongsheng Tong participates in the
writing and revision of articles. And Jianyou Shi, Lei Zhong guide the experimental ideas and methods.
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10.1002/cbdv.201900304
Chemistry & Biodiversity
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We deeply analyzed the crystal binding mode of GSK-3β small molecule inhibitor and GSK-3β protein, and obtained a superior core
structure combined with GSK-3β protein by computer virtual screening. We synthesized 17 unreported 3,5-diamino-N-substituted
benzamides and confirmed their structures by 1 H NMR, 13 C-NMR and HRMS. The in vitro antitumor activity of these compounds was
measured by MTT assay, and the structure-activity relationship was elucidated. The results showed that compound 4d had significant
antitumor activity against human colon cancer cells (HCT-116) with an IC 50 of 8.3 μM, and the selectivity for GSK-3β was commendable.
In addition, we also examined the induction of apoptosis by compound 4d and its in vivo pharmacokinetic properties.
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