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• With the increase of bacteria resistance, various drug- resistant bacteria are constantly being discovered. Methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), multi-drug resistant Escherichia coli, and multi-drug resistant Pseudomonas aeruginosa, causing lethal diseases worldwide and great difficulties in the treatment of community-acquired and nosocomial infections, ^[1~4] severely threated global public health and result in high economic costs.^[5] A possible solution for this fact is to research and develop novel antibiotics with new structure, target and mechanism of action for the unmet needs to control the infections caused by resistant bacteria, which always is the core of attention for medicinal chemists.^[6]

  Biphenyls are ubiquitous structure in natural products, such as terpenes, lignans, flavonoids, petides and alkaloids^[7~9] (Figure 1). These compounds exhibit broad-spec- trum biological activities, such as significantly reducing serum glutamic pyruvic transaminase, alleviating liver pathological damage, enhancing liver detoxification, and inhibiting human immunovirus (HIV) and tumor cells.^{[10, 11]} Biphenyls have attracted more and more attention in drugs, design and research because of their strong physiological activities. Many drugs with biphenyl structure have been developed, such as non-steroidal anti-inflammatory drugs felbinac, flurbiprofen, fenbufen, antipyretic analgesics diflunisal, and antibacterial drugs bifonazole^[12] (Figure 1).

  Figure 1

  

  Figure 1.  Structures of classical natural products and pharmaceuticals containing biphenyl fragment

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  Aminoguanidine derivatives, a kind of good metal anti-cancer and antiviral coordination complexes with low toxicity, have recently been investigated as antimicrobial agents.^[13-18] Wei et al.^[13] reported that a series of chalcone derivatives containing an aminoguanidine moiety (Compound I, Figure 2) showed potent antibacterial and antifungal activity against four gram-positive strains, four gram-negative strains and four clinical isolates of multidrug-resistant gram-positive bacterial strains with MICs in range of 1~16 μg/mL. To obtain more effective antibacterial agents with novel skeleton, in this study, the fragment-based design was reported using compound I as the lead compound, in which the modification was focused on changing the chalcone moiety to a biphenyl group and simultaneously introducing some F, Cl and Br substituents into the terminal phenyl ring. In this design, the two active fragments were hybridized into one molecule to expect a better activity. Thus, a series of new aminoguanidine derivatives containing a biphenyl moiety were synthesized, characterized and screened for their anti-bacterial and cytotoxicity.

  Figure 2

  

  Figure 2.  Lead compound and structure-based design of the target compounds

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  2.   Results and discussions

  2.1   Chemistry

  The synthetic route to prepare a new class of aminoguanidine-linked biphenyl derivatives from (4-formyl-phenyl) boronic acid and substituted bromobenzene is depicted in Scheme 1. The reaction of (4-formylphenyl)-boronic acid with substituted bromobenzene in the presence of potassium phosphate and palladium(Ⅱ) acetate in N, N- dimethylformamide-water condition produced 4'-sub- stituent-[1, 1'-biphenyl]-4-carbaldehydes (2a~2j) under the protection of nitrogen. Then target compounds 3a~3j were prepared by the condensation of 2a~2j with aminoguanidines. Finally, the structures of the target compounds were well characterized by ¹H NMR, ¹³C NMR, and high-resolu-tion mass spectrometry.

  Scheme 1

  

  Scheme 1.  Synthetic route of aminoguanidine-linked biphenyl derivatives

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  Taking compound 3a as an example in the structure confirmation. In the ¹H NMR spectrum, two singlets due to N—H of guanidyl were observed at δ 5.61 and 6.01, which were assigned to two NH₂ groups in terminal formed by the tautomerism of guanidyl group leading to the extension of conjugated chain. And the aromatic protons of terminal benzene ring were observed in δ 7.34~7.70. Two doublets (J＝8.4 Hz) due to aromatic protons of para-substituted phenyl ring were observed at δ 7.64 and 7.76. The absorption peak of C—H in imine was found at δ 8.04. The absorption peak in the hydrogen spectrum is completely in conformity with the hydrogen signal in the structure. ¹³C NMR spectra also give accurate information about the structure of the compound, which involved 10 kinds of carbon in different chemical environments. Moreover, the high-resolution mass spectrometry of 3a displayed an [M+H]⁺ signal at m/z 239.1291, which was corresponding to its molecular weight of 239.1295.

  2.2   Antimicrobial activity

  All of the target compounds 3a~3j were evaluated for their in vitro anti-bacterial activity using a serial dilution method to obtain the minimum inhibitory concentration (MIC) against five gram-positive strains (Staphylococcus aureus CMCC(B) 26003 and CMCC 25923, Streptococcus mutans BNCC 336931, Enterococcus faecalis CMCC 29212 and Bacillus subtilis CMCC 63501), and four gram-negative strains (Escherichia coli CMCC 25922 and CMCC 44568, and Pseudomonas aeruginosa CMCC 27853 and CMCC 10104) as well as two methicillin-resistant clinical isolates (Staphylococcus aureus ATCC 43300 and ATCC 33591). Gatifloxacin, moxifloxacin, norfloxacin, oxacillin, and penicillin were used as positive control drugs.

  The results of target compounds 3a~3j were described in Tables 1, 2 as MIC values against the Gram-positive and Gram-negative strains. It could be found that most of the tested compounds showed potent inhibitory effects against the strains with MICs in 0.5~8μg/mL, only compounds 3a, 3f, and 3h showed inhibitory activity at 128 μg/mL against S. aureus CMCC(B) 26003. Compound 3j showed the most potent inhibitory activity against S. aureus CMCC(B) 26003 and E. faecalis CMCC 29212 with a MIC value of 0.5 μg/mL, which was chosen to evaluate the antibacterial activity against two multidrug-resistant Gram-positive strains (S. aureus ATCC 43300 and ATCC 33591). As shown in Table 3, compound 3j also presented high activities (MIC＝4 or 0.5 μg/mL, respectively) against two multidrug-resistant S. aureus, which were slightly less active than gatifloxacin, moxifloxacin or norfloxacin (MIC＝0.5 or 0.25 μg/mL) but more potent than oxacillin (MIC＝64 or 8 μg/mL) and penicillin (MIC≥32 μg/mL).

  Table 1

  

  Table 1.  Inhibitory activity (MIC, μg•mL^－1) of compounds 3a~3j against Gram-positive bacteria

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  Compd.	R	Gram-positive strains
  		26003a	25923b	336931c	29212d	63501e
  3a	H	128	4	16	8	8
  3b	2-F	8	8	8	4	8
  3c	3-F	4	8	8	4	4
  3d	4-F	4	8	8	4	8
  3e	2-Cl	2	8	8	4	4
  3f	3-Cl	128	8	4	2	2
  3g	4-Cl	1	4	2	1	2
  3h	2-Br	128	4	8	4	4
  3i	3-Br	2	4	4	2	2
  3j	4-Br	0.5	4	1	0.5	1
  Gatifloxacin	—	0.125	0.125	1	1	2
  Moxifloxacin	—	0.125	0.125	0.5	1	2
  Norfloxacin	—	0.125	0.125	16	1	2
  Oxacillin	—	0.125	0.125	0.125	128	> 128
  Penicillin	—	0.125	0.125	0.125	128	128
  a Staphylococcus aureus CMCC(B)26003; b Staphylococcus aureus CMCC 25923; c Streptococcus mutans BNCC 336931; d Enterococcus faecalis CMCC 29212; e Bacillus subtilis CMCC 63501.

  Table 2

  

  Table 2.  Inhibitory activity (MIC, μg•mL^－1) of compounds 3a~3j against Gram-negative bacteria

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  Compd.	R	Gram-negative strains
  		25922a	44568b	27853c	10104d
  3a	H	16	16	16	16
  3b	2-F	8	8	16	8
  3c	3-F	8	8	16	16
  3d	4-F	16	16	16	16
  3e	2-Cl	8	8	16	8
  3f	3-Cl	4	8	8	8
  3g	4-Cl	4	8	8	4
  3h	2-Br	8	8	16	8
  3i	3-Br	4	4	16	4
  3j	4-Br	2	4	16	2
  Gatifloxacin	—	0.125	0.125	2	2
  Moxifloxacin	—	0.125	0.125	2	4
  Norfloxacin	—	0.125	0.125	2	4
  Oxacillin	—	128	> 128	> 128	128
  Penicillin	—	128	> 128	> 128	32
  a Escherichia coli CMCC 25922; b Escherichia coli CMCC 44568; c Pseudomonas aeruginosa CMCC 27853; dPseudomonas aeruginosa CMCC 10104.

  Table 3

  

  Table 3.  Inhibitory activity (MIC, μg•mL^－1) of compounds 3j against clinical isolates of multidrug-resistant strains

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  Compd.	R	Multidrug-resistant Gram-positive strains
  		43300a	33591b
  3j	4-Br	4	0.5
  Gatifloxacin	—	0.5	0.25
  Moxifloxacin	—	0.5	0.25
  Norfloxacin	—	0.5	0.25
  Oxacillin	—	64	8
  Penicillin	—	32	> 32
  a Staphylococcus aureus ATCC 43300; b Staphylococcus aureus ATCC 33591.

  In this study, some simple structure activity relationship patterns could be found between the antibacterial activity and the position and physicochemical properties of the different substituents on the phenyl ring. Generally, the halogen atom on different positions of phenyl ring played an advantageous role in increasing the antimicrobial activity relative to the non-substituted examples. A comparison of the halogen derivatives at the 4-position of the phenyl ring indicated that the different halogen atoms contributed to the antimicrobial activity in the order of Br > Cl > F. The position of the Br substituent on the phenyl ring also affected the activity of the compounds with an activity order of 4-Br > 3-Br > 2-Br.

  2.3   Cytotoxic activity

  The cytotoxic property of compound 3j was also investigated using CCK-8 test against one human normal cell lines HEK 293T. Interestingly, compound 3j showed low cytotoxic activity with an IC₅₀ value of 60.90 µmol/L. This result indicated that this compound has non-toxicity in normal cells at effective dose, suggesting a potential for a good therapeutic index when using as anti-bacterial agents.

  3.   Conclusions

  In the present work, a series of aminoguanidine derivatives containing a biphenyl moiety were synthesized, characterized, and screened for antimicrobial. Most of the tested compounds showed potent inhibitory activities against the selected strains with MICs in 0.5~8 μg/mL. Among of which, compound 3j showed the most potent inhibitory with a MIC value of 0.5 μg/mL against S. aureus CMCC(B) 26003 and E. faecalis CMCC 29212. Compound 3j also exhibited potent inhibition against two multidrug-resistant S. aureus ATCC 43300 and ATCC 33591 (MIC＝4 or 0.5 μg/mL, respectively), which were slightly less active than gatifloxacin, moxifloxacin and norfloxacin (MIC＝0.5 or 0.25 μg/mL) but much more potent than oxacillin (MIC＝64 or 8 μg/mL) and penicillin (MIC≥32 μg/mL). Furthermore, compound 3j also exhibited low degree of cytotoxicity. These findings strongly support the assumption that aminoguanidine-biphenyl conjugates could be used as a lead for further optimization of new antimicrobial agents to achieve promising therapeutics.

  4.   Experimental

  4.1   Instruments and reagents

  All of the reagents and solvents were purchased from Aladdin (Shanghai, China) or Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China), and were used as received. Melting points were determined in open capillary tubes and are uncorrected. Reaction courses were monitored by thin-layer chromatography on silica gel-precoated F₂₅₄ plates (Merck, Darmstadt, Germany). Developed plates were examined with UV lamps (254 nm). Nuclear magnetic resonance spectroscopy was performed on an AV-300 spectrometer (Bruker, Zurich, Switzerland) operating at 400 MHz for ¹H NMR and 100 MHz for ¹³C NMR, and using DMSO-d₆ as solvent and tetramethylsilane as the internal standard. Electrospray Ionization Mass Spectrometry (ESI-MS) experiments were performed on an IT-TOF mass spectrometer (Shimadzu, Tokyo, Japan) in negative ion mode.

  4.2   Synthesis method and spectral data

  4.2.1   Synthesis of 4'-substituent-[1, 1'-biphenyl]-4- carbaldehydes (2a~2j)s

  To a stirred solution of (4-formylphenyl) boronic acid (7.99 mmol, 1.5 equiv.) in N, N-dimethylformamide (DMF) (7 mL) and water (7 mL) was added substituted bromobenzene (5.35 mmol, 1 equiv.), K₃PO₄ (2.26 g, 10.67 mmol, 2 equiv.), and Pd(OAc)₂ (60 mg, 0.27 mmol, 0.05 equiv.) under N₂. The resulting solution was stirred overnight at room temperature before the addition of water (10 mL). The mixture was extracted with ethyl acetate (30 mL×2). The organic solution was dried over sodium sulfate, filtered and concentrated under vacuum. The crude residue was applied onto a silica gel column eluted with 1%~2% ethyl acetate in petroleum ether to afford 4'-substituent-(1, 1'- biphenyl)-4-carbaldehydes (2a~2j) as an off white solid (yield 18%~60%). The ¹H NMR characteristic spectra of intermediates 2a~2j were provided in the supporting information.

  4.2.2   Synthesis of 2-((4'-substituent-[1, 1'-biphenyl]-4-yl)methylene)hydrazine-1-carboximidamides (3a~3j)

  To a stirred solution of hydrazinecarboximidamide carbonate (353.88 mg, 2.6 mmol, 1.3 equiv.) in water (5 mL) was added NaOAc (213.2 mg, 2.6 mmol, 1.3 equiv.). After stirring for 0.5 h at room temperature, a mixture of 4'-substituent-(1, 1'-biphenyl)-4-carbaldehydes (2a~2j) (2 mmol, 1 equiv.) in EtOH (5 mL) was added. Then the resulting solution was stirred at 70 ℃ for 8 h. The reaction mixture was diluted with water (15 mL) and then cooled to room temperature. After stirred for 3 h, large amount of solids was precipitated. The solids were collected by filtration, washed with EtOH (0.4 mL×2), and then dried in an oven under reduced pressure to afford 2-((4'-substituent- (1, 1'-biphenyl)-4-yl)methylene)hydrazine-1-carboximida- mides (3a~3j) as a light yellow solid (yield 65%~93%).

  2-([1, 1'-Biphenyl]-4-yl)methylene)hydrazine-1-carboxi-midamide (3a): Light yellow solid, yield 93%. m.p. 211~213 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 5.61 (s, 2H, NH₂), 6.01 (s, 2H, NH₂), 7.34~7.70 (m, 5H, ArH), 7.64 (d, J＝8.4 Hz, 2H, ArH), 7.76 (d, J＝8.4 Hz, 2H, ArH), 8.04 (s, 1H, N＝CH); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 160.60, 142.80, 139.83, 139.25, 136.18, 128.98, 127.42, 126.82, 126.62, 126.48; MS m/z: 239 (M+1)⁺; ESI-HRMS calcd for C₁₄H₁₅N₄ ([M+H]⁺) 239.1291, found 239.1295.

  2-((2'-Fluoro-[1, 1'-biphenyl]-4-yl)methylene)hydra- zine-1-carboximidamide (3b): Light yellow solid, yield 86%. m.p. 203~206 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 5.65 (s, 2H, NH₂), 6.02 (s, 2H, NH₂), 7.29~7.58 (m, 6H, ArH), 7.79 (d, J＝8.3 Hz, 2H, ArH), 8.06 (s, 1H, N＝CH); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 161.21, 159.59 (d, ¹J_C—F＝218.6 Hz), 142.99, 137.04, 134.58, 131.03 (d, ⁴J_C—F＝3.1 Hz), 129.90 (d, ³J_C—F＝8.3 Hz), 129.23 (d, ⁴J_C—F＝2.8 Hz), 128.47 (d, ³J_C—F＝12.9 Hz), 126.81, 125.42 (d, ⁴J_C—F＝3.2 Hz), 116.61 (d, ²J_C—F＝22.5 Hz); MS m/z: 257 (M+1)⁺. ESI-HRMS calcd for C₁₄H₁₄FN₄ ([M+H]⁺) 257.1197, found 257.1195.

  2-((3'-Fluoro-[1, 1'-biphenyl]-4-yl)methylene)hydrazine- 1-carboximidamide (3c): Light yellow solid, yield 79%. m.p. 210~212 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 5.75 (s, 2H, NH₂), 6.10 (s, 2H, NH₂), 7.16~7.57 (m, 6H, ArH), 7.69 (d, J＝8.4 Hz, 2H, ArH), 7.79 (d, J＝8.4 Hz, 2H, ArH), 8.05 (s, 1H, N＝CH); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 163.22 (d, ¹J_C—F＝241.7 Hz), 160.99, 143.11, 142.73 (d, ³J_C—F＝7.7 Hz), 138.22 (d, ⁴J_C—F＝1.8 Hz), 137.16, 131.29 (d, ³J_C—F＝8.5 Hz), 127.28, 127.21, 122.96, 114.50 (d, ²J_C—F＝21.1 Hz), 113.59 (d, ²J_C—F＝21.8 Hz); MS m/z: 257 (M+1)⁺. ESI-HRMS calcd for C₁₄H₁₄FN₄ ([M+H]⁺) 257.1197, found 257.1191.

  2-((4'-Fluoro-[1, 1'-biphenyl]-4-yl)methylene)hydrazine- 1-carboximidamide (3d): Light yellow solid, yield 68%. m.p. 216~218 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 5.58 (s, 2H, NH₂), 5.99 (s, 2H, NH₂), 7.27~7.77 (m, 6H, ArH), 7.62 (d, J＝8.3 Hz, 2H, ArH), 7.79 (d, J＝8.3 Hz, 2H, ArH), 8.03 (s, 1H, N＝CH); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 161.84 (d, ¹J_C—F＝243.3 Hz), 160.62, 142.68, 138.14, 136.31 (d, ⁴J_C—F＝3.0 Hz), 136.17, 128.44 (d, ³J_C—F＝8.0 Hz), 126.79, 126.55, 115.73 (d, ²J_C—F＝21.0 Hz); MS m/z: 257 (M+1)⁺. ESI-HRMS calcd for C₁₄H₁₄FN₄ ([M+H]⁺) 257.1197, found 257.1201.

  2-((2'-Chloro-[1, 1'-biphenyl]-4-yl)methylene)hydrazine- 1-carboximidamide (3e): Light yellow solid, yield 73%. m.p. 226~228 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 5.59 (s, 2H, NH₂), 5.99 (s, 2H, NH₂), 7.38~7.58 (m, 6H, ArH), 7.69 (d, J＝8.2 Hz, 2H, ArH), 7.77 (d, J＝8.4 Hz, 2H, ArH), 8.05 (s, 1H, N＝CH); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 160.74, 142.53, 139.58, 137.76, 136.49, 131.41, 131.25, 129.91, 129.27, 029.09, 127.54, 125.93. MS m/z: 273 (M+1)⁺. ESI-HRMS calcd for C₁₄H₁₄ClN₄ ([M+H]⁺) 273.0902, found 273.0900.

  2-((3'-Chloro-[1, 1'-biphenyl]-4-yl)methylene)hydrazine- 1-carboximidamide (3f): Light yellow solid, yield 65%. m.p. 208~210 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 5.69 (s, 2H, NH₂), 6.06 (s, 2H, NH₂), 7.39~7.79 (m, 8H, ArH), 8.04 (s, 1H, N＝CH); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 160.63, 142.58, 141.98, 137.56, 136.80, 133.80, 130.77, 127.19, 126.84, 126.80, 126.14, 125.16; MS m/z: 273 (M+1)⁺. ESI-HRMS calcd for C₁₄H₁₅ClN₄ ([M+H]⁺) 273.0902, found 273.0905.

  2-((4'-Chloro-[1, 1'-biphenyl]-4-yl)methylene)hydrazine- 1-carboximidamide (3g): Light yellow solid, yield 70%. m.p. 197~200 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 5.64 (s, 2H, NH₂), 6.03 (s, 2H, NH₂), 7.51 (d, J＝8.6 Hz, 2H, ArH), 7.65 (d, J＝8.4 Hz, 2H, ArH), 7.66 (d, J＝8.6 Hz, 2H, ArH), 7.78 (d, J＝8.40 Hz, 2H, ArH), 8.03 (s, 1H, N＝CH); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 160.63, 142.65, 138.62, 137.81, 136.53, 132.22, 128.91, 128.22, 126.86, 126.58; MS m/z: 273 (M+1)⁺. ESI-HRMS calcd for C₁₄H₁₄ClN₄ ([M+H]⁺) 273.0902, found 273.0909.

  2-((2'-Bromo-[1, 1'-biphenyl]-4-yl)methylene)hydrazine- 1-carboximidamide (3h): Light yellow solid, yield 78%. m.p. 218~219 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 5.79 (s, 2H, NH₂), 6.09 (s, 2H, NH₂), 7.29~7.77 (m, 8H, ArH), 8.05 (s, 1H, N＝CH); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 160.43, 142.63, 141.62, 139.66, 136.28, 133.07, 131.38, 129.34, 129.26, 128.04, 125.93, 121.71; MS m/z: 317 (M+1)⁺. ESI-HRMS calcd for C₁₄H₁₄BrN₄ ([M+H]⁺) 317.0396, found 317.0389.

  2-((3'-Bromo-[1, 1'-biphenyl]-4-yl)methylene)hydrazine- 1-carboximidamide (3i): Light yellow solid, yield 69%. m.p. 206~208 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 5.63 (s, 2H, NH₂), 6.03 (s, 2H, NH₂), 7.40~7.88 (m, 8H, ArH), 8.03 (s, 1H, N＝CH); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 160.70, 142.57, 142.28, 137.49, 136.85, 131.09, 130.12, 128.99, 126.84, 126.82, 125.58, 122.47; MS m/z: 317 (M+1)⁺. ESI-HRMS calcd for C₁₄H₁₄BrN₄ ([M+H]⁺) 317.0396, found 317.0391.

  2-((4'-Bromo-[1, 1'-biphenyl]-4-yl) methylene)hydrazine- 1-carboximidamide (3j): Light yellow solid, yield 80%. m.p. 219~221 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 5.81 (s, 2H, NH₂), 6.11 (s, 2H, NH₂), 7.62~7.67 (m, 6H, ArH), 7.78 (d, J＝8.3 Hz, 2H, ArH), 8.04 (s, 1H, N＝CH); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 160.36, 142.69, 138.94, 137.91, 136.41, 131.80, 128.53, 126.89, 126.51, 120.80; MS m/z: 317 (M+1)⁺. ESI-HRMS calcd for C₁₄H₁₄BrN₄ ([M+H]⁺) 317.0396, found 317.0398.

  4.3   Evaluation of anti-bacterial activity in vitro

  The anti-bacterial activity in vitro against S. aureus CMCC(B) 26003 and CMCC 25923, S. pyogenes CMCC 32067, E. faecalis CMCC 29212, B. subtilis CMCC 63501; E. coli CMCC 25922 and CMCC 44568, P. aeruginosa CMCC 27853 and CMCC 10104, as well as two methicillin-resistant clinical isolates (S. aureus ATCC 43300 and ATCC 33591) was evaluated using a two-fold serial dilution technique, and the final concentrations of compounds obtained were in the range of 0.5~128 μg/mL. Test bacteria were grown to mid-log phase in Mueller-Hinton Broth (MHB) or Tryptone Soya Broth (TSB), and diluted 1000-fold in the same medium. The bacteria of 10⁵ CFU/mL were inoculated into MHB or TSB and dispensed at 0.2 mL/well in a 96-well microtiter plate. As positive controls, gatifloxacin, moxifloxacin, norfloxacin, oxacillin, and penicillin were used. Test compounds were prepared in DMSO, the final concentration of which did not exceed 0.05%. The MIC was defined as the concentration of a test compound that completely inhibited bacteria growth during 24 h incubation at 37 ℃. Bacteria growth was determined by measuring the absorption at 630 nm using a microtiter enzyme-linked immunosorbent assay (ELISA) reader. All experiments were carried out three times.

  4.4   Evaluation of cytotoxicity in vitro

  Human embryonic kidney 293T cells (HEK 293T cells) were used to test the anticancer activity of the new compounds. HEK 293T cells were grown in Dulbecco modified Eagle medium supplemented with fetal bovine serum (10%), and antibiotics (penicillin-streptomycin mixture (100 U/mL)). Cells at 80% to 90% confluence were split by trypsin (0.25% in PBS; pH 7.4), and the medium was changed at 24-h intervals. The cells were cultured at 37 ℃ in a 5% CO₂ incubator. The cells were grown to three passages, and approximately 1×10⁴ cells were seeded into each well of a 96-well plate and allowed to incubate to allow attachment of the cells to the substrate. After 24 h, the medium was replaced with DMEM supplemented with 10% FBS containing various concentrations (0.3, 1, 3, 10, 30, and 100 μmol/L) of test compounds and incubated for 48 h. For each concentration, three wells were set in parallel. Then, 20 µL of CCK-8 solution was added to each well. After incubation for 3 h, the optical density was measured at 450 nm using a microtiter ELISA reader. The IC₅₀ values were defined as the concentrations inhibiting 50% of cell growth.

  Supporting Information The ¹H NMR spectra of intermediates 2a~2j, and ¹H NMR, ¹³C NMR spectra and high resolution mass spectrum of target compounds 3a~3j. The Supporting Information is available free of charge via the Internet at http://sioc-journal.cn/.

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  Figure 1  Structures of classical natural products and pharmaceuticals containing biphenyl fragment

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  Figure 2  Lead compound and structure-based design of the target compounds

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  Scheme 1  Synthetic route of aminoguanidine-linked biphenyl derivatives

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  Table 1.  Inhibitory activity (MIC, μg•mL^－1) of compounds 3a~3j against Gram-positive bacteria

  Compd.	R	Gram-positive strains
  		26003a	25923b	336931c	29212d	63501e
  3a	H	128	4	16	8	8
  3b	2-F	8	8	8	4	8
  3c	3-F	4	8	8	4	4
  3d	4-F	4	8	8	4	8
  3e	2-Cl	2	8	8	4	4
  3f	3-Cl	128	8	4	2	2
  3g	4-Cl	1	4	2	1	2
  3h	2-Br	128	4	8	4	4
  3i	3-Br	2	4	4	2	2
  3j	4-Br	0.5	4	1	0.5	1
  Gatifloxacin	—	0.125	0.125	1	1	2
  Moxifloxacin	—	0.125	0.125	0.5	1	2
  Norfloxacin	—	0.125	0.125	16	1	2
  Oxacillin	—	0.125	0.125	0.125	128	> 128
  Penicillin	—	0.125	0.125	0.125	128	128
  a Staphylococcus aureus CMCC(B)26003; b Staphylococcus aureus CMCC 25923; c Streptococcus mutans BNCC 336931; d Enterococcus faecalis CMCC 29212; e Bacillus subtilis CMCC 63501.

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  Table 2.  Inhibitory activity (MIC, μg•mL^－1) of compounds 3a~3j against Gram-negative bacteria

  Compd.	R	Gram-negative strains
  		25922a	44568b	27853c	10104d
  3a	H	16	16	16	16
  3b	2-F	8	8	16	8
  3c	3-F	8	8	16	16
  3d	4-F	16	16	16	16
  3e	2-Cl	8	8	16	8
  3f	3-Cl	4	8	8	8
  3g	4-Cl	4	8	8	4
  3h	2-Br	8	8	16	8
  3i	3-Br	4	4	16	4
  3j	4-Br	2	4	16	2
  Gatifloxacin	—	0.125	0.125	2	2
  Moxifloxacin	—	0.125	0.125	2	4
  Norfloxacin	—	0.125	0.125	2	4
  Oxacillin	—	128	> 128	> 128	128
  Penicillin	—	128	> 128	> 128	32
  a Escherichia coli CMCC 25922; b Escherichia coli CMCC 44568; c Pseudomonas aeruginosa CMCC 27853; dPseudomonas aeruginosa CMCC 10104.

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  Table 3.  Inhibitory activity (MIC, μg•mL^－1) of compounds 3j against clinical isolates of multidrug-resistant strains

  Compd.	R	Multidrug-resistant Gram-positive strains
  		43300a	33591b
  3j	4-Br	4	0.5
  Gatifloxacin	—	0.5	0.25
  Moxifloxacin	—	0.5	0.25
  Norfloxacin	—	0.5	0.25
  Oxacillin	—	64	8
  Penicillin	—	32	> 32
  a Staphylococcus aureus ATCC 43300; b Staphylococcus aureus ATCC 33591.

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