HETEROCYCLES

Short Paper | Regular issue | Vol. 83, No. 8, 2011, pp. 1843-1851
Received, 26th March, 2011, Accepted, 12th May, 2011, Published online, 23rd May, 2011.
DOI: 10.3987/COM-11-12218

■ Synthesis of the Identical Linker Mode Twin-Drug Type C2-Symmetrical Molecules

Fumiko Fujisaki, Haruka Usami, Saya Nakashima, Shiomi Iwashita, Yurie Kurose, Nobuhiro Kashige, Fumio Miake, and Kunihiro Sumoto*

Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan

Abstract

In connection with our studies on antibacterial active compounds against gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) strains, some molecular modifications were attempted. In this study, molecular transformations of aminoguanidines and related amines to the represented C2-symmetrical molecules (Aa–b, Ba–b, and Ca–b) were investigated. In addition, some C3-symmetrical compounds (Da–b) were also prepared.

INTRODUCTION
With reference to work on new antibacterial compounds, extensive efforts have been made to find new promising candidates. Many reports on molecular recognition properties of symmetrical macromolecules have appeared,1 and it is well known that many receptors or membrane proteins in the native state often have a high order of symmetrical interface. In the infection process by bacteria or virus, microorganisms usually use sugar-binding proteins such as lectins.2 We have been interested in compounds that interfere with such a recognition process in order to find new leads for antibacterial agents.3 Since molecular recognition via complexes of C2- or C3-symmetry (2-fold or 3-fold symmetrical geometry) is a common feature of several important receptors,4 we have designed twin-drug type symmetrical molecules in connection with our studies on antibacterial compounds.5 In this article, syntheses of target designed C2- or C3-symmetrical molecules in the search for biologically active leads are described.

DESIGN OF TARGET SYMMETRICAL MOLECULE
In terms of molecular symmetry, small symmetrical molecules frequently appear in various synthetic twin-drug type molecules5 and biologically active compounds. Biologically active C2-symmetrical molecules are usually constructed on a symmetrical alkyl linker or another symmetrical aromatic template.5,6 For linker mode twin-drug symmetrical molecules, the nature of a linker plays an important role in binding to the receptor site for biological activity.5,6 It was thought worthwhile to undertake a synthetic molecular modification study with the aim of obtaining new canditates with antibacterial activity. From this point of view, molecular modification of aminoguanidines seemed to be interesting because some aminoguanidine derivatives are known to interfere with the cross-linking of sugar chains.7 Furthermore, a number of bacteria utilize heparan sulfate proteoglycans such as syndecans as attachment factors for host epithelia for coursing infections in many body sites. Heparan sulfate sugar chains have a repeated sulfated structure of sugar units.8 These facts encouraged us to design C2-symmetrical structures having two guanidine groups in the target molecules, because a guanidine functionality (a guanidinium ion structure in physiological conditions) is expected to interfere with anionic sulfate moieties of heparan sulfate sugar chains by charge-charge (ionic) interactions.5 We therefore carried out synthetic investigation of new C2-symmetrical derivatives (Aa, and Ab) (Figure 1). Molecular modification of the represented C2-symmetrical molecules (A) can be considered to be an identical twin-drug type approach utilizing a biphenyl scaffold as a linker.

We also designed C2-symmetrical molecules represented as structure B or C (Figure 1). In addition, we prepared C3-symmetrical cis-1,3,5-trisubstituted molecules Da and Db (Figure 2) from the reaction of 1,3,5-cis-cyclohexanetricarbonyl chloride9 and corresponding aminoguanidines for bioassay and comparison of antibacterial activity with that of C2-symmetrical target molecules. These modifications to C3-symmetrical molecules can also be considered to be an identical triplet-drug type approach in the search for bioactive leads.

CHEMISTRY AND RESULTS
Synthesis of identical linker mode twin-drug type C2-symmetrical molecules (A, B, and C) could be achieved by condensation reaction of the corresponding dicarbonyl chloride as templates and aminoguanidines or corresponding amines. The C2-symmetrical structures of the synthesized compounds were easily confirmed by 13C-NMR spectroscopic analysis. All of the twin-drug type compounds showed magnetically equivalent spectroscopic signal patterns (appearance of signals assignable to half of the symmetrical molecules), indicating a C2-symmetrical molecular feature in solution (see Experimental). Symmetrical target compound (Ba) could be obtained from the reaction of isophthaloyl chloride with aminoguanidine.

TLC analysis of the reaction products of isophthaloyl chloride with aminoguanidine indicated the formation of three types of derivatives (Figure 3). Compound Ba was easily dehydrated to give cyclized heterocyclic compounds (Bc and Bd), and the ratio of the three products was dependent on the reaction conditions. At an elevated reaction temperature, a condensed target derivative (Ba) further proceeded the intramolecular dehydration reaction of both acyl aminoguanidines in the molecule, and compound Bd was obtained as a sole product. At a lower reaction temperature, a monodehydrated unsymmetrical product (Bc) could be isolated (see Experimental). By NMR spectroscopic analysis of signal patterns, compound Bd also showed a C2-symmetrical molecular feature in solution (see Experimental). C3-symmetrical compounds (Da and Db) were also prepared from the reaction of 1,3,5-cis-cyclohexanetricarbonyl chloride with corresponding amines. The C3-synmetrical structures of the products were easily confirmed by 13C-NMR spectroscopic analysis (see Experimental data).
A bioassay for antibacterial activity [determination of the minimum concentrations (MICs) of the compounds] was carried out by authentic methods according to the Japanese Society of Chemotherapy. Most of the synthesized C2- or C3-symmetrical molecules showed no remarkable antibacterial activity against either Escherichia coli (gram-negative) or Staphylococcus aureus (gram-positive). However, two compounds (Aa and Ab) that used biphenyl as a linker showed significant antibacterial activity (MIC = 0.330 and 0.225 µM/mL, respectively) against E. coli but did not show remarkable antibacterial activity against S. aureus strain.
Further synthetic applications on the basis of the above chemical and biological information for related symmetrical derivatives, particularly in the search for biological active lead compounds, are under investigation.

EXPERIMENTAL
Melting points are uncorrected. IR spectra were measured by a Shimadzu FT/IR-8100 spectrometer. The 1H- and 13C-NMR spectra were obtained by a JEOL JNM A-500 at 35 °C. The chemical shifts were expressed in δ ppm downfield from an internal tetramethylsilane (TMS) signal. The signal assignments were confirmed by 1H – 1H two-dimensional (2D) correlation spectroscopy (COSY), 1H – 13C heteronuclear multiple quantum coherence (HMQC), and 1H – 13C heteronuclear multiple-bond connectivity (HMBC) spectra. High FAB-MS spectra were obtained by a JEOL JMS-HX110 mass spectrometer.
Assays for Antibacterial Activity
We used S. aureus ATCC6538P and E. coli NBRC14237 (NIHJ) (gram-positive and gram-negative bacteria, respectively) as target organisms. Synthesized compounds (A, B, C, and D) were dissolved in dimethyl sulfoxide (DMSO) to a concentration of 1.280 g/mL. The minimum inhibitory concentration (MIC) of a standard strain was measured by the authentic microdilution method to monitor the bacterial growth turbidity in Muller-Hinton broth according to the Japanese Society of Chemotherapy.10,11
2,2'-(4, 4’-Biphenyl-1,1’-dicarbonyl)bis(hydrazinecarboximidamide) Dihydrochloride (Aa)
A mixture of biphenyl-4,4'-dicarbonyl dichloride (0.50 g, 1.79 mmol) and aminoguanidine hydrochloride (0.79 g, 7.14 mmol) in a round-bottomed flask was heated slowly up to 200 °C in an oil bath and kept for 10 min. After cooling, the mixture was triturated with water. Insoluble material Aa (0.53 g, 69.3%) was collected by filtration. Mp >200 °C (dec). IR (KBr) cm-1: 3372, 3293, 3211, 3139, 1660, 1626, 1597. FAB-MS (positive) m/z: 355 (M+H)+. 1H-NMR (DMSO-d6) δ: 7.4–8.2 (8H, br, C=(N+H2)NH2), 7.92 (4H, d, J = 8.5 Hz, Ar H-3, H-5, H-3’, H-5’), 8.07 (4H, d, J = 8.5 Hz, Ar H-2, H-6, H-2’, H-6’), 9.75 (2H, br, CONHNH), 10.75 (2H, br, CONHNH), 13C-NMR (DMSO-d6) δ: 126.7 (Ar C-3, C-5, C-3’, C-5’), 128.6 (Ar C-2, C-6, C-2’, C-6’), 131.2 (Ar C-1, C-1’), 142.3 (Ar C-4, C-4’), 158.8 (NHC(=NH)NH2), 166.0 (CO). Anal. Calcd for C16H18N8O2 • 2HCl: C, 44.97; H, 4.72; N, 26.22. Found: C, 44.70; H, 4.77; N, 26.50.
N'4,N'4'-Bis(4,5-dihydro-1H-imidazol-2-yl)biphenyl-4,4'-dicarbohydrazide Dihydribromide (Ab)
A mixture of biphenyl-4,4'-dicarbonyl dichloride (0.80 g, 2.86 mmol) and 2-hydrazinoimidazoline hydrobromide (1.14 g, 6.30 mmol) in a round-bottomed flask was heated slowly up to 190 °C in an oil bath and kept for 40 min. After cooling, the precipitated material was recrystallized from water to give Ab dihydrobromide (0.88 g, 54.0%). The mother liquor was made alkaline with K2CO3 and the precipitated material was filtered to give an Ab free base. To a solution of this free base in MeOH (10 mL) was added a solution of 10% hydrochloride in MeOH (4 mL). Concentration of the solvent gave Ab hydrochloride (0.48 g, 35.0%, mp >230 °C). Total yield was 89.0%. The following data was shown as dihydrobromide. Mp >230 °C. IR (KBr) cm-1: 3181, 1683, 1652. FAB-MS (positive) m/z: 407 (M+H)+. 1H-NMR (DMSO-d6) δ: 3.70 (8H, s, imidazole H-4, H-5), 7.94 (4H, d, J = 8.5 Hz, Ar H-3, H-5, H-3’, H-5’), 8.06 (4H, d, J = 8.5 Hz, Ar H-2, H-6, H-2’, H-6’), 8.2–9.2 (4H, br, imidazole -NH-C=N+H-), 10.40 (2H, br, CONHNH), 10.92 (2H, br, CONHNH), 13C-NMR (DMSO-d6) δ: 42.8 (imidazole C-4, C-5), 126.8 (Ar C-3, C-5, C-3’, C-5’), 128.6 (Ar C-2, C-6, C-2’, C-6’), 131.0 (Ar C-1, C-1’), 142.4 (Ar C-4, C-4’), 160.9 (imidazole C-2), 166.0 (CO). Anal. Calcd for C20H22N8O2 • 2HBr: C, 42.27; H, 4.26; N, 19.72. Found: C, 42.35; H, 4.22; N, 19.66.
Reaction of Isophthaloyl Dichloride with Aminoguanidine Hydrochloride:
Formation of (Ba) and (Bc)
A mixture of isophthaloyl chloride (0.50 g, 2.46 mmol) and aminoguanidine hydrochloride (0.66 g, 5.97 mmol) in a round-bottomed flask was heated slowly up to 177 °C in an oil bath and kept for 10 min. After cooling, EtOH (5 mL) was added to the reaction mixture and insoluble material Ba (0.44 g, 50.9%) was collected by filtration. The filtrate was concentrated in vacuo and the residue was recrystallized from EtOH to give Bc (0.10 g, 12.2%).
2,2'-(1,3-Phenylenedicarbonyl)bis(hydrazinecarboximidamide) Dihydrochloride (Ba)
Mp 233 °C (dec). IR (KBr) cm-1: 1658. FAB-MS (positive) m/z: 279 (M+H)+. 1H-NMR (DMSO-d6) δ: 7.64–7.67 (1H, m, Ar H-5), 7.7–8.0 {8H, br, C=(N+H2)NH2}, 8.15–8.16 (2H, m, Ar H-4, H-6), 8.61 (1H, s, Ar H-2), 9.81 (2H, s, CONHNH), 10.87 (2H, br, CONHNH), 13C-NMR (DMSO-d6) δ: 127.9 (Ar C-2), 128.3 (Ar C-5), 131.3 (Ar C-4, C-6), 131.9 (Ar C-1, C-3), 158.8 {NHC(=NH)NH}, 165.9 (CO). Anal. Calcd for C10H14N8O2 • 2HCl • H2O: C, 32.53; H, 4.91; N, 30.35. Found: C, 32.50; H, 4.84; N, 30.36.
2-(3-(5-Amino-4H-1,2,4-triazol-3-yl)phenylcarbonyl)hydrazinecarboximidamide Dihydrochloride (Bc)
Mp >240 °C (EtOH). IR (KBr) cm-1: 1688, 1670, 1651. FAB-MS (positive) m/z: 261 (M+H)+. 1H-NMR (DMSO-d6) δ: 7.2–8.2 [8H, m, {C=(N+H2)NH2} + {triazole –NH + -N+H3}], 7.67 (1H, t, J = 7.9 Hz, Ar H-5), 8.08 (1H, t, J = 7.9 Hz, Ar H-6), 8.15–8.19 (1H, m, Ar H-4), 8.55 (1H, br s, Ar H-2), 9.81 (1H, br s, CONHNH), 10.84 (1H, br s, CONHNH), 13C-NMR (DMSO-d6) δ: 125.7 (Ar C-2), 126.6 (Ar C-3), 129.0 (Ar C-5), 129.3 (Ar C-4), 129.6 (C-6), 132.6 (Ar C-1), 149.8 (triazole C-3), 153.1 (triazole C-5), 158.8 {NHC(=NH)NH}, 165.9 (CO). Anal. Calcd for C10H12N8O • 2HCl • 1.4H2O: C, 33.51; H, 4.72; N, 31.26. Found: C, 33.43; H, 4.61; N, 31.43.
N'1,N'3-Bis(4,5-dihydro-1H-imidazol-2-yl)benzene-1,3-dicarbohydrazide Dihydrobromide (Bb)
A mixture of isophthaloyl chloride (0.50 g, 2.46 mmol) and 2-hydrazinoimidazoline hydrobromide (1.06 g, 5.86 mmol) in a round-bottomed flask was heated slowly up to 220 °C in an oil bath and kept for 3 min. After cooling, the precipitate material was recrystallized from MeOH (5 mL) to give Bb (0.66 g, 54.5%). Mp >245 °C (dec). IR (KBr) cm-1: 3227, 3154, 1690, 1658, 1618. FAB-MS (positive) m/z: 331 (M+H)+. 1H-NMR (DMSO-d6) δ: 3.69 (8H, s, imidazole H-4, H-5), 7.69 (1H, t, J = 7.9 Hz, Ar H-5), 8.14–8.16 (2H, m, Ar H-4, H-6), 8.52 (1H, s, Ar H-2), 8.69 (4H, br, imidazole -NH-C=N+H-), 10.46 (2H, s, CONHNH), 10.92 (2H, s, CONHNH), 13C-NMR (DMSO-d6) δ: 42.8 (imidazole C-4, C-5), 127.7 (Ar C-2), 128.4 (Ar C-5), 131.3 (Ar C-4, C-6), 131.9 (Ar C-1, C-3), 160.8 (imidazole C-2), 165.8 (CO). Anal. Calcd for C14H18N8O2 • 2HBr • 1.6 H2O: C, 32.28; H, 4.49; N, 21.51. Found: C, 32.29; H, 4.68; N, 21.64.
3,3'-(1,3-Phenylene)bis(4H-1,2,4-triazol-5-amine) Dihydrochloride (Bd)
A mixture of isophthaloyl chloride (0.69 g, 3.40 mmol) and aminoguanidine hydrochloride (0.83 g, 7.51 mmol) in a round-bottomed flask was heated slowly up to 210 —240 °C in an oil bath and kept for 3.5 h. After cooling, MeOH (30 mL) was added to the reaction mixture and separated insoluble material was collected by filtration. The solid material was recrystallized from c-HCl. The crystal material was dissolved in water and the aqueous solution was made alkaline with K2CO3. The precipitated material was filtered and purified by silica gel column chromatography with EtOH-28% ammonia solution (100 : 3) as a solvent to afford Bd as a free base. Treatment with 10% methanolic hydrochloride (4 mL) gave Bd dihydrochloride (0.45 g, 42.1%). Mp >240 °C (dec). IR (KBr) cm-1: 1689. FAB-MS (positive) m/z: 243 (M+H)+. 1H-NMR (DMSO-d6) δ: 7.0–9.0 (8H, br, NH + -N+H3), 7.72 (1H, t, J = 7.9 Hz, Ar H-5), 8.13 (2H, dd, J = 7.9, 1.8 Hz, Ar H-4, H-6), 8.52 (1H, t, J = 1.8 Hz, Ar H-2). 13C-NMR (DMSO-d6) δ: 123.3 (Ar C-2), 126.8 (Ar C-1, C-3), 128.0 (Ar C-4, C-6), 129.8 (Ar C-5), 149.1 (triazole C-3), 152.7 (triazole C-5). Anal. Calcd for C10H10N8 • 2HCl • 0.2H2O: C, 37.68; H, 3.92; N, 35.15. Found: C, 37.81; H, 3.87; N, 34.87.
2,2'-(1,3-Pyridinedicarbonyl)bis(hydrazinecarboximidamide) Dihydrochloride (Ca)
A mixture of pyridine-2,6-dicarbonyl dichloride (0.50 g, 2.45 mmol) and aminoguanidine hydrochloride (0.67 g, 6.06 mmol) in a round-bottomed flask was heated slowly up to 145 °C in an oil bath and kept for 3 min. After cooling, the reaction mixture was triturated with water and concentrated in vacuo. The solid residue was recrystallized from MeOH to give Ca (0.24 g, 27.9%). Mp 240 °C (dec). IR (KBr) cm-1: 3364, 3154, 1682. FAB-MS (positive) m/z: 280 (M+H)+. 1H-NMR (DMSO-d6) δ: 7.5–8.0 {8H, br, C=(N+H2)NH2}, 8.27–8.28 (3H, m, Pyridine H-3, H-4, H-5), 10.07 (2H, s, CONHNH), 11.38 (2H, s, CONHNH), 13C-NMR (DMSO-d6) δ: 125.3 (Pyridine C-3, C-5), 139.6 (Pyridine C-4), 147.4 (Pyridine C-2, C-6), 158.9 {NHC(=NH)NH}, 163.0 (CO). Anal. Calcd for C9H13N9O2 • 2HCl • 1.5H2O: C, 28.51; H, 4.78; N, 33.24. Found: C, 28.40; H, 4.55; N, 33.17.
N'2,N'6-Bis(4,5-dihydro-1H-imidazol-2-yl)pyridine-2,6-dicarbohydrazide
Hydrobromide • Hydrochloride (Cb)
A solution of pyridine-2,6-dicarbonyl dichloride (0.50 g, 2.45 mmol) and 2-hydrazinoimidazoline hydrobromide (1.06 g, 5.86 mmol) in DMF (1 mL) was stirred at 130 °C for 3 min under an N2 stream. After cooling, precipitated material was washed with AcOEt and dissolved in water (1 mL). Addition of EtOH (1 mL) and isoPrOH (2 mL) to the solution gave Cb (0.62 g, 53.9%). Mp >240 °C. IR (KBr) cm-1: 3165, 1702, 1655, 1604. FAB-MS (positive) m/z: 332 (M+H)+. 1H-NMR (DMSO-d6) δ: 3.70 (8H, br s, imidazole H-4, H-5), 8.29 (3H, s, Pyridine H-3, H-4, H-5), 8.7 (4H, br, imidazole -NH-C=N+H-), 10.67 (2H, br s, CONHNH), 11.57 (2H, s, CONHNH), 13C-NMR (DMSO-d6) δ: 42.8 (imidazole C-4, C-5), 125.5 (pyridine C-3, -5), 139.8 (Pyridine C-4), 147.2 (Pyridine C-2, C-6), 160.8 (imidazole C-2), 162.8 (CO). Anal. Calcd for C13H17N9O2 • 1.5HBr • 0.5HCl • 1.5H2O: C, 31.36; H, 4.45; N, 25.32. Found: C, 31.32; H, 4.64; N, 25.05.
2, 2’, 2’’-(1,3,5-Cylohexanetricarbonyl)tris(carboximidamide) Trihydrochloride (Da)
A mixture of 1,3,5-cis-cyclohexanetricarbonyl chloride9 (0.50 g, 1.84 mmol) and aminoguanidine hydrochloride (0.73 g, 6.60 mol) in a round-bottomed flask was heated slowly up to 165 °C in an oil bath and kept for 5 min. After cooling, the precipitated solid material was washed with MeOH/EtOH to give Da (0.53 g, 58.5%). Mp 248–250 °C (dec). IR (KBr) cm-1: 3363, 3141, 1662. FAB-MS (positive) m/z: 385 (M+H)+. 1H-NMR (DMSO-d6) δ: 1.40–1.48 (3H, dd, J = 15.5, 12.5 Hz, Cyclehexane HA-2, HA-4, HA-6), 2.12 (3H, d, J =12.5 Hz, Cyclehexane HB-2, HB-4, HB-6), 2.50–2.51 (3H, m, Cyclohexane H-1, H-3, H-5), 7.58–7.69 {12H, br, C=(N+H2)NH2}, 9.58 (3H, s, CONHNH), 10.21 (3H, s, CONHNH), 13C-NMR (DMSO-d6) δ: 30.0 (Cyclohexane C-2, C-4, C-6), 40.4 (Cyclohexane C-1, C-3, C-5), 158.6 {NHC(=NH)NH}, 174.1 (CO). Anal. Calcd for C12H24N12O3 • 3 HCl • H2O: C, 28.16; H, 5.71; N, 32.84. Found: C, 28.25; H, 5.71; N, 32.86.
N'1,N'3,N'5-Tris(4,5-dihydro-1H-imidazol-2-yl)cyclohexane-1,3,5-tricarbohydrazide
Trihydrobromide (Db)
A mixture of 1,3,5-cis-cyclohexanetricarbonyl chloride9 (0.30 g, 1.10 mmol) and 2-hydrazinoimidazoline hydrobromide (0.66 g, 3.65 mmol) in a round-bottomed flask was heated slowly up to 180 °C in an oil bath and kept for 10 min. After cooling, the resulting mixture was washed with EtOH/isoPrOH to give Db (0.31 g, 39.8%). Mp >250 °C. IR (KBr) cm-1: 3179, 1712, 1645. FAB-MS (positive) m/z: 463 (M+H)+. 1H-NMR (DMSO-d6) δ: 1.41–1.49 (3H, m, Cyclehexane ring HA-2, HA-4, HA-6), 2.12–2.14 (3H, m, Cyclehexane ring HB-2, HB-4, HB-6), 2.31–2.37 (3H, m, Cyclehexane ring H-1, H-3, H-5), 3.64 (12H, s, imidazole H-4, H-5), 8.50 (6H, br, imidazole -NH-C=N+H-), 10.26 (3H, s, CONHNH), 10.40 (3H, s, CONHNH), 13C-NMR (DMSO-d6) δ: 30.0 (Cyclehexane ring C-2, C-4, C-6), 40.4 (Cyclehexane ring C-1, C-3, C-5), 42.7 (imidazole C-4, C-5), 160.7 (imidazole C-2), 173.9 (CO). Anal. Calcd for C18H30N12O3 • 3 HBr: C, 30.66; H, 4.72; N, 23.83. Found: C, 30.82; H, 4.87; N, 23.90.

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