SYNTHESIS OF SUBSTRATES SPECIFIC FOR HUMAN SPLEEN FIBRINOLYTIC PROTEINASE (SFP)

Two peptide anilides, Suc-Tyr-Leu-Val-pNA (III) and Ac-Ser-His-I.eu-Val-pNA (V) were synthesized by the conventional method with the object of obtaining specific substrates for human spleen fibrinolytic proteinase (SFP). The K_cat/K_m values for hydrolysis of HI and V by SFP were 22 64 7 and 2 700, respectively. On the contrary, the K_cat/K_m values for hydrolysis of III and V by porcine pancreatic elastase were 21 and 9, respectively.     

(Z)-Dimethylamino-l-(4-bromophenyl)-l-(3-pyridyl) propene (H 102/09), a New Selective Inhibitor of the Neuronal 5-Hydroxytryptamine Uptake

Abstract The inhibition of the uptake of ³H–(–)–noradrenaline (NA), ³H–dopamine and ¹⁴C–5–hydroxytryptamine (5–HT) in mouse brain slices by (Z)–3–dimethylamino–l–(4–bromophenyl)–l–(3–pyridyl)propene (H 102/09), desipramine and chlorimipramine and their releasing effect on the ³H–amines previously accumulated in the slices were examined. The interactions with reserpine produced hypothermia and sedation and the 5–hydroxytryptophan (5–HTP) syndrome in mice were also studied. Due to the poor inhibitory activity on the NA uptake H 102/09 was a more selective inhibitor of the 5–HT uptake than was chlorimipramine, particularly after administration in vivo, where it was as potent as chlorimipramine (ED50 = 19 μmol/kg intraperitoneally). In vitro chlorimipramine was 6 to 12 times more active than H 102/09. Desipramine was a very selective inhibitor of the NA uptake in vitro and in vivo. The compounds were generally more potent in inhibiting the uptake than in releasing the amines. However, in striatal slices the inhibition of DA uptake could be due to the releasing effect since the difference in potencies were small. The effect of desipramine on 5–HT uptake and that of H 102/09 on NA uptake could also involve a release component. The 5–HTP syndrome was potentiated by H 102/09 and chlorimipramine but not by desipramine. The reserpine hypothermia but not the sedation was potently antagonized and reversed by desipramine and by chlorimipramine at high doses but not by H 102/09, suggesting that NA but not 5–HT is involved in the hypothermic action of reserpine.     

Detection of human neutrophil elastase with peptide‐bound cross‐linked ethoxylate acrylate resin analogs

Abstract: An assessment of elastase‐substrate kinetics and adsorption at the solid–liquid interface of peptide‐bound resin was made in an approach to the solid‐phase detection of human neutrophil elastase (HNE), which is found in high concentration in chronic wound fluid. N‐succinyl‐alanine‐alanine‐proline‐valine‐p‐nitroanilide (suc‐Ala‐Ala‐Pro‐Val‐pNA), a chromogenic HNE substrate, was attached to glycine‐cross‐linked ethoxylate acrylate resins (Gly‐CLEAR) by a carbodiimide reaction. To assess the enzyme‐substrate reaction in a two‐phase system, the kinetic profile of resin‐bound peptide substrate hydrolysis by HNE was obtained. A glycine and di‐glycine spacer was placed between the resin polymer and substrate to assess the steric and spatial requirements of resin to substrate with enzyme hydrolysis. The enzymatic activities of suc‐Ala‐Ala‐Pro‐Val‐pNA and suc‐Ala‐Ala‐Pro‐Ala‐pNA on the solid‐phase resin were compared with similar analogs in solution. An increase in visible wavelength absorbance was observed with increasing amounts of substrate‐resin and enzyme concentration. Enzyme hydrolysis of the resin‐bound substrate was also demonstrated on a polypropylene surface, which was employed for visible absorbance of released chromophore. A soluble active substrate analog was released from the resin through saponification of the ethoxylate ester linkages in the resin polymer. The resin‐released conjugate of the HNE substrate demonstrated an increased dose response with increasing enzyme concentration. The synthesis and assay of elastase substrates bound to CLEAR resin gives an understanding of substrate‐elastase adsorption and activity at the resin's solid–liquid interface for HNE detection with a solid‐phase peptide.     

Inhibitory effect of 5-fluorouracil on human cytochrome P(450) isoforms in human liver microsomes

Objective A number of case reports have been described regarding drug interactions with 5-fluorouracil (5-FU) and co-administered drugs. However, little is known regarding the inhibitory potential of 5-FU on the metabolism of co-administered drugs by cytochrome P₄₅₀ (CYP). The aim of the present study was to elucidate the inhibitory effect of 5-FU on CYP isoforms using human liver microsomes.Methods The inhibitory effect of 5-FU on CYP1A2, CYP2C9, CYP2C19, CYP2C8, CYP2E1, CYP2D6, and CYP3A4 activities was examined with specific probe drugs in human liver microsomes.Results 5-FU showed little or no inhibitory effect on CYP-catalyzed reactions in human liver microsomal preparations.Conclusion 5-FU has no inhibitory effect on CYP isoforms or drug metabolism causing drug interaction with 5-FU. The mechanism that causes drug interaction between co-administered drugs and 5-FU may not be related to direct CYP inhibition by 5-FU.     

Modulating human proteinase activated receptor 2 with a novel antagonist (GB88) and agonist (GB110)

BACKGROUND AND PURPOSE

Many cells express proteinase activated receptor 2 (PAR2) on their plasma membrane. PAR2 is activated by proteolytic enzymes, such as trypsin and tryptase that cleave the receptor N-terminus, inititating signalling to intracellular G proteins. Studies on PAR2 have relied heavily upon activating effects of proteases and peptide agonists that lack stability and bioavailability in vivo.

EXPERIMENTAL APPROACH

A novel small molecule agonist GB110 and an antagonist GB88 were characterized in vitro against trypsin, peptide agonists, PAR2 antibody, PAR1 agonists and flow cytometry,in seven cell lines using intracellular Ca²⁺ mobilization and examined in vivo against PAR2- and PAR1-induced rat paw oedema.

KEY RESULTS

GB110 is a potent non-peptidic agonist activating PAR2-mediated Ca²⁺ release in HT29 cells (EC₅₀∼200 nM) and six other human cell lines, inducing PAR2 internalization. GB88 is a unique PAR2 antagonist, inhibiting PAR2 activated Ca²⁺ release (IC₅₀∼2 µM) induced by native (trypsin) or synthetic peptide and non-peptide agonists. GB88 was a competitive and surmountable antagonist of agonist 2f-LIGRLO-NH₂, a competitive but insurmountable antagonist of agonist GB110, and a non-competitive insurmountable antagonist of trypsin. GB88 was orally active and anti-inflammatory in vivo, inhibiting acute rat paw oedema elicited by agonist GB110 and proteolytic or peptide agonists of PAR2 but not by corresponding agonists of PAR1 or PAR4.

CONCLUSIONS AND IMPLICATIONS

The novel PAR2 agonist and antagonist modulate intracellular Ca²⁺ and rat paw oedema, providing novel molecular tools for examining PAR2-mediated diseases.     

Synthesis of plasmin substrates and relationship between their structure and plasmin activity

The plasmin substrates, D-Ile-Phe-Lys-pNA (I), 3-MV-Phe-Lys-pNA (II), Ile-Phe-Lys-pNA (III), D-Pro-Phe-Lys-pNA (IV), CP-Phe-Lys-pNA (V) and Pro-Phe-Lys-pNA (VI), were synthesized by the conventional solution method and the kinetic parameters of their amidolysis by plasmin were determined. It was found that the free amino group of the D-amino acid in substrates (I) and (IV) made a contribution to an increment in affinity between the substrate and plasmin.     

Assessment of the sensitizing potential of textile disperse dyes and some of their metabolites by the loose-fit coculture-based sensitization assay (LCSA)

Certain textile disperse dyes are known to cause allergic reactions of the human skin. Here, we examined 8 disperse dyes and 7 products of azo-cleavage of these dyes in an in vitro assay. We used the loose-fit coculture-based sensitization assay (LCSA) of primary human keratinocytes and of allogenic dendritic cell-related cells for combined testing of the sensitizing and irritative properties of these substances. The obtained data were compared to data generated in a modified version of the local lymph node assay by our working group. Disperse Blue 1 (DB1), p-nitroaniline (pNA) and p-aminoacetanilide (AAA) showed no sensitizing potential under our experimental conditions. Disperse Blue 124 (DB124), Disperse Yellow 3 (DY3), Disperse Orange 37/76 (DO37), Disperse Blue 106 (DB106), Disperse Red 1 (DR1), 2-amino-p-cresol (ApC), Disperse Orange 3 (DO3) and 2,6-dichloro-4-nitroaniline (DCh) were categorized as extreme sensitizers. Para-phenylenediamine (pPD) was categorized as strong sensitizer, and 2-amino-5-nitrothiazole (ANT) and 2-(N-ethylanilino)-ethanol (EAE) as weak sensitizers. All dyes, except for DB1, and ApC turned out to be strong irritants. DB1, ANT and DCh showed only weak irritative potential. PPD, pNA, EAE and AAA did not show any irritative effect at the concentration range tested. These results correlate with data derived from the modified version of LLNA and human data. Therefore, the LCSA represents a suitable test system to simultaneously analyse two crucial properties of substances relevant for allergy induction.     

Comparison of the Effects of a New Vasodilator Pinacidil and Nifedipine on Isolated Blood Vessels

Abstract: In isolated human crural veins studied in vitro pinacidil (0.038–380 μM) caused a concentration-related inhibition of noradrenaline, 18 μM (NA) and 127 mM K⁺-induced contractions. Pinacidil was more potent in inhibiting the NA-contraction than that induced by K⁺, whereas the reverse was seen for nifedipine. At the highest concentrations greater inhibitions of the NA-induced contractions could be obtained with pinacidil than with nifedipine. The inhibitory effect of pinacidil on the K⁺-induced contractions was eliminated during a 1 hr wash-out period. In contrast to this, the inhibitory effect of nifedipine could not be eliminated during 4 hrs repeated wash-out. Pinacidil was completely devoid of inhibitory effect on ⁴⁵Ca net influx in rat aorta, whereas nifedipine caused a significant reduction of influx. The results indicate that both pinacidil and nifedipine are effective vasodilatators in human vessels. Pinacidil seems to be more effective in NA-induced contractions than does nifedipine. The mechanism of action of pinacidil cannot be attributed to an inhibitory effect on cellular calcium entry.     

Effect of p-chlorophenylalanine on the loss and maintenance of tolerance to ethanol

Rats were rendered tolerant to the motor-impairing effects of ethanol by daily oral administration. Subsequently, ethanol was withdrawn and the effect of p-chlorophenylalanine (p-CPA) on tolerance loss was examined. In two separate studies it was demonstrated that p-CPA, in a dosage regimen that produces extensive depletion of brain serotonin (5-HT), accelerated tolerance loss. These experiments suggest that at least part of p-CPA's inhibitory effect on net tolerance development to ethanol can be accounted for by its accelerating effect on tolerance loss; however, an inhibitory effect on tolerance acquisition cannot be excluded. On the other hand, once tolerance was established, p-CPA did not affect the maintenance of tolerance to ethanol.     

Four Weeks’Inhalation Exposure of Rats to p-Cymene Affects Regional and Synaptosomal Neurochemistry

Abstract: Long-lasting effects of inhalation exposure to p-cymene (p-isopropyl-toluene; CAS No. 99-87-6) on regional and subcellular brain neurochemistry were studied. Male Long-Evans rats were exposed to 0, 50, or 250 p.p.m. p-cymene 6 hr/day, 5 days/week for four weeks followed by an exposure-free period of 8 weeks. Synaptosomes were isolated from whole brain minus cerebellum and used as an ex situ model for in situ conditions at the level of the presynaptic nerve terminal. There was no persistent effect on wet weight (regional) or regional noradrenaline (NA), dopamine (DA), or 5-hydroxytryptamine (5-HT) concentrations owing to exposure. Yield of synaptosomal protein was statistically significantly reduced in an exposure concentration-related manner (Control: 16.6±3.1; 50 p.p.m.: 9.2±2.1; 250 p.p.m.: 8.6±1.7 mg protein/g tissue, mean± 1 S.D.). Synaptosomal NA and DA concentrations and acethycholinesterase, butyrylcholinesterase, and lactate dehydrogenase activities were statistically significantly increased when expressed relative to synaptosomal protein. It is hypothesized that a reduced density and number of synapses in situ are functionally compensated for by increased NA and DA release from noradrenergic and dopaminergic presynaptic nerve terminals. The applicability of the synaptosome as an ex situ neurochemical research model for the presynaptic CNS nerve terminal in situ for the study of solvent neurotoxicity in rats was further supported.     

中国黑眼镜蛇毒蛋白酶natrahagin抑制血小板聚集和动脉血栓形成的作用

研究从中国黑眼镜蛇毒中纯化的中国黑眼镜蛇毒蛋白酶natrahagin对血小板聚集 ,血浆纤维蛋白原水平和动脉血栓形成的影响 .采用比浊法测定兔血小板聚集率 ,双缩脲法测定血浆纤维蛋白原浓度 .应用胰蛋白酶损伤血管内皮的方法制作家兔颈动脉血栓模型评价natrahagin抑制血栓形成的作用 .结果发现 ,新西兰家兔natrahagin ( 0 .0 2 5～ 0 .1mg·kg- 1,iv)剂量依赖性地抑制二磷酸腺苷 ( 10 μmol·L- 1)和胶原 ( 10 0mg·L- 1)诱导的血小板聚集 ,显著降低血浆纤维蛋白原水平 .剂量为 0 .1mg·kg- 1时 ,血浆纤维蛋白原水平降低 33.3% .并能有效地抑制兔颈动脉血栓的形成 ,效应呈剂量依赖性 .0 .1mg·kg- 1剂量组对血栓形成的抑制率达到 4 5.4 % .研究提示 ,natrahagin抑制动脉血栓形成的作用与其抑制纤维蛋白原介导的血小板聚集和降低血浆纤维蛋白原水平的作用有关     

Combined effect of cadmium (CdCl2) and high temperature on HeLa S3 cells

The combined effect of cadmium (CdCl₂) and high temperature on the subsequent growth, the synthesis of nucleic acids and protein, and the cell cycle in HeLa S₃ cells was investigated. The subsequent growth rate was depressed by cadmium at 37.1°C. This suppression effect was enhanced at 40.4° C compared with the effect at 37.1° C. The synthesis of DNA, RNA and protein was inhibited at higher temperatures in cultivation without cadmium, and depressed at each temperature in a concentration-dependent manner by the addition of cadmium. The DNA, RNA and protein-IC₅₀ values at 40.4° C decreased compared with the values at 37.1° C. The DNA and RNA-IC₅₀ values were significantly decreased (p<0.01,p<0.005, respectively) depending on the temperature. After treating the cells with cadmium at 40.4° C, the DNA/BrdUrd distribution showed that the rate of dead cells increased and the rate of the G₁/G₀ phase decreased. These results indicate that the inhibitory effect of cadmium on the subsequent growth of HeLa S₃ cells is enhanced at high temperature and this enhancement is related to the increased inhibitory effect of cadmium on DNA and RNA synthesis at high temperature.     

Membrane interaction of synthetic peptides related to the putative fusogenic region of PH-30α, a protein in sperm-egg fusion

In order to investigate the relationship between structure and function of a putative fusogenic region of PH-30a, a protein active in sperm-egg fusion, two peptides, SFP22 and SFP23, whose sequences correspond to the residues 90-111 and 89-111 of PH-30α, respectively, were chemically synthesized. An analog of SFP23, SFP23AA, which has an Ala-Ala sequence instead of the Pro-Pro sequence in SFP23, was also prepared. The CD study indicated that SFP22 and SFP23 mainly took a β-structure in the presence of DPPC and DPPC/DPPG (3/1) vesicles, while SFP23AA showed an α-helical pattern though the a-helical content calculated was low (25–30%). α-Helical CD curve was observed for these peptides in trifluoroethanol. The membrane-perturbing activity of SFP22 and SFP23 was weaker than that of SFP23AA. On the other hand, the membrane-fusogenic activity of SFP22 and SFP23 to acidic phospholipid bilayers was much stronger than that of SFP23AA. All the peptides caused very weak cell lysis. These results are consistent with the reported speculation [Blobel, C. P. et al. (1992), Nature (London) 356, 248–252 that residues 90–111 of PH-30α may be the fusogenic region and suggest that the Pro-Pro sequence is one of the important factors for holding the active secondary structure of the fusogenic region of PH-30α in membranes.     

In vitro Antiviral Effects and 3D QSAR Study of Resveratrol Derivatives as Potent Inhibitors of Influenza H1N1 Neuraminidase

The anti-influenza virus activities of 50 resveratrol (RV: 3, 5, 4′-trihydroxy-trans-stilbene) derivatives were evaluated using a neuraminidase (NA) activity assay. The results showed that 35 compounds exerted an inhibitory effect on the NA activity of the influenza virus strain A/PR/8/34 (H1N1) with 50% inhibitory concentration (IC₅₀) values ranging from 3.56 to 186.1 μm . Next, the 35 RV derivatives were used to develop 3D quantitative structure–activity relationship (3D QSAR) models for understanding the chemical–biological interactions governing their activities against NA. The comparative molecular field analysis (CoMFA r² = 0.973, q² = 0.620, q_test² = 0.661) and the comparative molecular similarity indices analysis (CoMSIA r² = 0.956, q² = 0.610, q_test² = 0.531) were applied. Afterward, molecular docking was performed to study the molecular interactions between the RV derivatives and NA. Finally, a cytopathic effect (CPE) reduction assay was used to evaluate the antiviral effects of the RV derivatives in vitro. Time-of-addition studies demonstrated that the RV derivatives might have a direct effect on viral particle infectivity. Our results indicate that the RV derivatives are potentially useful antiviral compounds for new drug design and development for influenza treatment.     

The effect of silymarin (Silybum marianum) on human skin fibroblasts in an in vitro wound healing model

Context: Silymarin, a flavonolignan from Silybum marianum (L.) Gaertn. (Asteraceae), has been reported to have antioxidant and anti-inflammatory properties. Therefore, it may be worthwhile to study the effect of silymarin on wound healing.

Objective: To evaluate the effect of silymarin on human fibroblast cells in an in vitro model of wound healing.

Materials and methods: Human fibroblast cells were treated with different concentrations (4.5, 9, 18, 36 µg/mL) of silymarin. The effects of silymarin on cell viability, proliferation, collagen synthesis, and expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthetase (iNOS) were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5-bromo-2′-deoxy-uridine, hydroxyproline analysis and real-time PCR, respectively. The effect of silymarin on cellular antioxidant status was determined by protection against hydrogen peroxide (H₂O₂)-induced cell injury and free radical scavenging activity (ABTS assay) of the cells.

Results: Results of the present study indicate that pretreatment of fibroblast cells with silymarin significantly protected cells against H₂O₂-induced injury (p < 0.05). After an 18?h treatment of cells with 36 µg/mL silymarin, total antioxidant capacity of cells significantly increased (p < 0.05). Furthermore, pretreatment of human fibroblast cells with silymarin significantly inhibited lipopolysaccharide (LPS)-induced COX-2 mRNA expression (p < 0.001). There was no significant difference in fibroblast proliferation and collagen synthesis between treatment and control groups (p > 0.05).

Discussion and conclusion: Silymarin may be useful as a therapeutic agent for the treatment of cutaneous wounds through its antioxidation and anti-inflammation effects.     

Effect of atropine and acetylcholine on nerve stimulated output of noradrenaline and dopamine-beta-hydroxylase from isolated rabbit and guinea pig hearts

Summary The role of a cholinergic muscarinic inhibitory mechanism in sympathetic neurotransmission was investigated in isolated rabbit and guinea pig hearts with intact sympathetic nerves. The effect of varying frequencies of stimulation (2.5, 5 and 10 Hz) on the concentration of noradrenaline (NA) and dopaminebeta-hydroxylase (DBH) released into the perfusate was investigated. Stimulation in the presence of atropine sulfate (3.4 M) resulted in an augmented outflow of NA at all three frequencies while DBH outflow was significantly increased only at 5 and 10 Hz. d-Tubocurarine (2.0 M) attenuated the augmenting effect of atropine on NA release at all frequencies of stimulation whereas it negated the significant effect of atropine on DBH release. Nerve stimulation in the presence of acetylcholine (0.55 M) resulted in a significant decrease in the concentrations of NA and DBH in the perfusate.It is suggested that atropine augments NA outflow in part by blocking an intrinsic muscarinic inhibitory mechanism. Acetylcholine's inhibitory effect on NA release is reflected in a similar decrease in DBH release and, therefore, may function in vivo via an effect on exocytosis at the adrenergic nerve ending.Supported by NIH Training Grant GM 01417.Part of a thesis submitted in partial fulfillment for the degree of Doctor of Philosophy in Pharmacology from The Ohio State University.     

Antiplatelet aggregation activity of compounds isolated from Guttiferae species in human whole blood

Twenty compounds isolated from Calophyllum inophyllum L., C. inophylloides King, Garcinia opaca King, G. bancana Miq., and G. parvifolia Miq. (Guttiferae) were evaluated for their ability to inhibit platelet aggregation in human whole blood induced by arachidonic acid (AA), collagen, and adenosine diphosphate (ADP). The compounds inhibited platelet aggregation in a dose-dependent manner. Among the compounds tested, 2-(3-methylbut-2-enyl)-1,3,5-trihydroxyxanthone and 2-(3-methylbut-2-enyl)-1,3,5,6- tetrahydroxyxanthone showed strong inhibitory activity on platelet aggregation induced by AA with IC₅₀ values of 115.9 and 113.0?μM, respectively. Rubraxanthone showed inhibitory activity against aggregation caused by the three inducers, and was the most effective antiplatelet compound against collagen-induced platelet aggregation with an IC₅₀ value of 47.0?μM. Macluraxanthone, GB-1a, pyranoamentoflavone, and a neoflavonoid showed selective inhibitory activity on platelet aggregation induced by ADP.     

Muscarinic modulation of endogenous noradrenaline release from adrenergic terminals in the guinea-pig colon

1 The present study examined the role of muscarinic receptors in the modulation of noradrenaline (NA) release in the guinea-pig isolated distal colon. The spontaneous endogenous NA overflow assayed by HPLC-ED was taken as an index of NA release from enteric noradrenergic nerve terminals. 2 Physostigmine (10 μm ) significantly enhanced spontaneous endogenous NA overflow. Hyoscine (muscarinic antagonist), (R)-(-)-trihexyphenidyl and telenzepine (M₁-selective antagonists), and 11[[2-[(diethylamino)methyl]-1-piperydil]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116, M₂-selective antagonist) inhibited NA overflow in a concentration dependent manner, with the following EC₅₀ values: 131.74 (18.19–953.96), 101.62 (58.83–175.60), 150 (60–330), 30 (5–170) nm , respectively. 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, M₁- and M₃- selective antagonist) had no significant effect up to 100 μm . 3 The muscarinic agonist oxotremorine inhibited NA overflow in a concentration dependent manner, with an EC₅₀ value of 0.67 (0.30–1.51) μm . The response to oxotremorine was inhibited by muscarinic antagonists with the following order of potency: hyoscine = (R)-(-)-trihexyphenidyl = telenzepine > 4-DAMP >> AF-DX 116. 4 In the presence of 3 μm tetrodotoxin (TTX), the effect of oxotremorine and 4-DAMP was unchanged, while hyoscine, (R)-(-)-trihexyphenidyl, telenzepine and AF-DX 116, instead of inhibiting, significantly enhanced NA overflow. 5 The present results indicate that, in the guinea-pig colon, endogenous acetylcholine sustains spontaneous NA release by activating muscarinic receptors possibly located on interneurones. In addition, inhibitory muscarinic receptors may exist on adrenergic terminals.     

肿瘤的化学治疗ⅩⅩⅩⅣ.与对-双(2-氯乙基)氨基-ω-溴代苯乙酮-六甲撑四胺复合物(AT-584)有关的研究——Ⅲ.对-[双(2-氯乙基)氨甲基]-ω-溴代苯乙酮-六甲撑四胺复合物的合成

In a previous paper of this series we reported that the hexamethylenetetramine salt of p-bis-(2-chloroethyl)-amino-ω-bromoacetophenone (Ⅰ, AT-584) had pronounced inhibitory action with long-acting efficacy on the growth of several experimental tumoturs, and that the antiturnout property was due to the mustard group-and the long-acting efficacy was probably related to the quarternary structure. Since nitrogen mustards of aliphatic type usually possess more effective action than those of aromatic ones, the hexamethylenetetramine salt of p-[bis-(2-chloroethyl)-aminomethyl]-ω-bromoacetophenone (Ⅱ) was synthesized.The starting material for the synthesis of compound Ⅱ was p-[bis-(2-chloroethyl) aminomethyl]-benzoic acid hydrochloride (Ⅷ), which was prepared from ethyl p-toluate or p-tolunitrile via bromination with NBS, condensation with diethanolamine, Chlorination with thionyl chloride and hydrolysis with hydrochloric acid successively. Gompound Ⅷ was chlorinated by thionyl chloride in dry benzene to give its acid chloride (Ⅸ), which without being isolated, was treated with diazomethane to yield the corresponding diazoketone(Ⅹ). The latter, without being isolated in pure state was decomposed in dioxane by hydrobromic acid to give p-[bis (2-chloroethyl) aminomethyl]-ω-bromoacetophenone (Ⅺ). Finally, the desired product (Ⅱ) was obtained by treating Ⅺ with hexamethylenetetramine in chloroform.Preliminary pharmacological tests showed that compound Ⅱ did not possess significant inhibitory action against sarcoma 180 in mice.