The syntheses and protein linking properties of succinimidyl 4-hydrazinobenzoate hydrochloride (SHBH) and succinimidyl 6-hydrazinonicotinate hydrochloride (SHNH), two new heterobifunctional linkers which lead to hydrazino-modified proteins, are described. SHBH-modified proteins are unstable due to the presence of the phenylhydrazine moiety. This problem was overcome by synthesizing the hydrazinopyridine analogue SHNH, and the conjugates derived from this linker are stable. Tc(V) oxo precursors readily add to hydrazinopyridine-modified proteins to yield the desired 99mTc-radiolabeled protein. 99mTc-hydrazinopyridine-polyclonal IgG conjugates are useful agents for the imaging of focal sites of infection.

The design and synthesis of hydrazinopyridine bifunctional chelating agents (BCA's) featuring amide, ester, and disulfide groups are described. The BCA's site-specifically react with the free thiol groups of the tumor-specific monoclonal antibody fragment C46.3 using a one-pot in situ reduction and conjugation procedure from the F(ab')2 to give Fab'-linker conjugates. Molar substitution ratios (MSR's) of the hydrazinopyridine conjugates were comparable to the theoretical (maximum) number of thiols per fragment determined by free hydrazine and residual thiol assays. The series of C46.3 Fab'-linker conjugates were 99mTc-labeled in greater than 95% radiochemical purity by incubation with 99mTc-tricine for 1 h at room temperature. In order to evaluate the conjugates for radiopharmaceutical applications, the tumor localization and biodistribution properties of the radiolabeled Fab'-linker conjugates, compared to the direct labeled fragment, were tested in nude mice bearing LS174T xenografts. Depending upon the structure of the linker connecting the radiolabeled hydrazinopyridine group to the antibody fragment, we observed a variation in kidney uptake and whole-body clearance. Diester- and monoester-linked conjugates exhibited lower kidney uptake and faster whole-body clearance than the corresponding linker containing amide groups. This result may be interpreted as evidence for rapid metabolism of ester compared to amide groups in the kidney following uptake. At 24-h postinjection, the monoester-linked conjugate 99mTc-C46.3 Fab'-BA displayed the highest tumor: blood ratio (16.2) compared to the directly labeled conjugate (6.6) and is therefore a potential clinical candidate for imaging breast and ovarian cancer.

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Anew limonoid, mzikonol [2], in addition to the known limonoids mzikonone [1], azadirone [3], 1,2-dihydroazadirone [4], and nimbolinin B [6], was isolated from the root bark of Turraea robusta. In addition, a new tetranortriterpene lactone, turranolide [51, and the known triterpene, butyrospermol [7], both possible protolimonoids, were isolated.Compounds were identified by chemical and spectroscopic methods.Turraea robusta Guerke (Meliaceae) is a small tree found in East Africa. The Zaramo tribe of Tanzania utilizes a tea prepared from the root bark of this tree, called mzikoziko in the Zaramo language, as a traditional medicine for the treatment of stomach pains and diarrhea. The leaves are used as an antidote for poisoning (1). We reported earlier the isolation and identification of a new compound, mzikonone [1], as the major tetranortriterpene (limonoid) from the MeOH extract of the root bark (2). Limonoids from two other members of this genus, Turrea floribunda and Turrea obtusifolia, have been reported (3). The latter plant was shown to contain prieurianin, while the former con-