Paradoxical activation of Raf by a novel Raf inhibitor.

BACKGROUND: Raf is a proto-oncogene that is activated in response to growth factors or phorbol esters, and is thought to activate MAP kinase kinase-1 (MKK1) and hence the classical MAP kinase (MAPK) cascade. RESULTS: The compound ZM 336372 is identified as a potent and specific inhibitor of Raf isoforms in vitro. Paradoxically, exposure of cells to ZM 336372 induces > 100-fold activation of c-Raf (measured in the absence of compound), but without triggering any activation of MKK1 or p42 MAPK/ERK2. The ZM 336372-induced activation of c-Raf occurs without any increase in the GTP-loading of Ras and is not prevented by inhibition of the MAPK cascade, protein kinase C or phosphatidylinositide 3-kinase. ZM 336372 does not prevent growth factor or phorbol ester induced activation of MKK1 or p42 MAPK/ERK2, or reverse the phenotype of Ras- or Raf-transformed cell lines. The only other protein kinase inhibited by ZM 336372 out of 20 tested was SAPK2/p38. Although ZM 336372 is structurally unrelated to SB 203580, a potent inhibitor of SAPK2/p38, the mutation of Thr106-->Met made SAPK2/p38 insensitive to ZM 336372 as well as to SB 203580. CONCLUSIONS: Raf appears to suppress its own activation by a novel feedback loop, such that inhibition is always counterbalanced by reactivation. These observations imply that some agonists reported to trigger the cellular activation of c-Raf might actually be inhibitors of this enzyme, and that compounds which inhibit the kinase activity of Raf might not be useful as anticancer drugs. The binding sites for ZM 336372 and SB 203580 on Raf and SAPK2/p38 are likely to overlap.

Cellular pharmacology and in vivo activity of a new anticancer agent, ZD9331: a water-soluble, nonpolyglutamatable, quinazoline-based inhibitor of thymidylate synthase.

ZD9331 is a drug that was developed from a potent class of water-soluble, C7-methyl-substituted, quinazoline-based inhibitors of thymidylate synthase (TS) that are transported into cells via a saturable, carrier-mediated system (reduced folate carrier, or RFC) but are not substrates for folylpolyglutamate synthetase. ZD9331 is the gamma-tetrazole analogue of 2-desamino-2, 7-dimethyl-N10-propargyl-2'fluoro-5,8-dideaza folate (ZM214888), with a TS Ki of approximately 0.4 nM. ZD9331 exhibits potent growth inhibitory and cytotoxic activity; e.g., IC50 for the inhibition of human W1L2 lymphoblastoid cell line was 7 nM. The addition of thymidine to the culture medium increased the IC50 in W1L2 cells >10, 000-fold, demonstrating the high specificity of the drug for TS. ZD9331 is transported into cells predominantly via the RFC. Accordingly, it competes with methotrexate (MTX) and folinic acid for cellular uptake and has reduced activity against two cell lines with low expression of the RFC (L1210:1565 and CEM/MTX). In addition, a cell line with acquired resistance to ZD9331 displays reduced uptake of both ZD9331 and MTX. A mouse cell line (L1210:RD1694), with acquired resistance to ZD1694 due to reduced folylpolyglutamate synthetase activity, was not significantly cross-resistant to ZD9331. The flux through TS, as measured by 3H release from 5-[3H]deoxyuridine, was rapidly inhibited when cells were incubated with ZD9331. However, because ZD9331 cannot form polyglutamates, TS activity recovered rapidly once cells were placed in drug-free medium. The minimum curative dose of ZD9331 in the i.m. L5178Y TK-/- tumor model was approximately 3 mg/kg when given by 24-h continuous infusion, and it was 25-50 mg/kg when given by a single i.p. or i.v. injection. ZD9331 had antitumor activity against the L5178Y TK+/- tumor when administered by 7-day continuous infusion; growth delays of more than 5 days (and some cures) were seen at doses of 25-50 mg/kg/day. At higher doses, significant weight loss (gastrointestinal toxicity) and myelosuppression (neutropenia and thrombocytopenia) were observed, suggesting that these may be dose-limiting toxicities in the Phase I clinical studies.

ZD1694 (Tomudex): a new thymidylate synthase inhibitor with activity in colorectal cancer.

ZD1694 (Tomudex) is a new antifolate which is a specific inhibitor of thymidylate synthase (TS). Evidence suggests that ZD1694 has a spectrum of activity that only partially overlaps with 5-fluorouracil (modulated with leucovorin) against colon tumours in vitro. Potent cytotoxic activity is dependent upon active uptake into cells via the reduced folate/methotrexate cell membrane carrier (RFC) and subsequent metabolism to polyglutamated forms (tri, tetra and pentaglutamates). These polyglutamates are approximately 60-fold more active as TS inhibitors and are not effluxed readily from cells. Extensive polyglutamation also occurs in various mouse tissues (e.g. small intestinal epithelium, liver and kidney), resulting in high tissue/plasma drug ratios which persist for a prolonged period. ZD1694 has antitumour activity in mice, although the high plasma thymidine in this species complicates: (1) the interpretation of therapeutic index; (2) tumour types in which activity is likely to be observed; and (3) translation of doses and schedules for clinical evaluation. ZD1694 entered clinical study and has completed Phase I and II evaluation, with activity observed in several tumour types. Appreciable activity in the Phase II colorectal study (29% objective response rate on interim analysis) led to the current Phase III study, randomised against 5-fluorouracil/leucovorin.

Quinazoline antifolate thymidylate synthase inhibitors: gamma-linked L-D, D-D, and D-L dipeptide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583).

The syntheses of gamma-linked L-D, D-D, and D-L dipeptide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) are described. The general methodology for the synthesis of these molecules involved the preparation of the dipeptide derivatives employing solution phase peptide synthesis followed by condensation of the dipeptide free bases with the appropriate pteroic acid analogue via diethyl cyanophosphoridate (DEPC) activation. In the final step, tert-butyl esters were removed by trifluoroacetic acid (TFA) hydrolysis. Z-L-Glu-OBut-gamma-D-Ala-OBut, for example, was prepared from alpha-tert-butyl N-(benzyloxycarbonyl)-L-glutamate and tert-butyl D-alaninate via isobutyl-mixed anhydride coupling. The Z-group was removed by catalytic hydrogenolysis and the resulting dipeptide free base condensed with 2-desamino-2-methyl-N10-propargyl-5,8-dideazapteroic acid via DEPC coupling. Finally, tert-butyl esters were removed by TFA hydrolysis to give ICI 198583-gamma-D-Ala. The compounds were tested as inhibitors of thymidylate synthase and L1210 cell growth. Good enzyme and growth inhibitory activity were found with gamma-linked L-D dipeptides, the best examples being the Glu-gamma-D-Glu derivative 35 (Ki = 0.19 nM, L1210 IC50 = 0.20 +/- 0.017 microM) and the Glu-gamma-D-alpha-aminoadipate derivative 39 (Ki = 0.12 nM, L1210 IC50 = 0.13 +/- 0.063 microM). In addition, ICI 198583 L-gamma-D-linked dipeptides were resistant to enzymatic degradation in mice.

Design and synthesis of Cyclopenta[g]quinazoline-based antifolates as inhibitors of thymidylate synthase and potential antitumor agents(,).

Following the development of raltitrexed, the synthesis of nonpolyglutamatable inhibitors of TS that do not use the reduced folate carrier (RFC) for cellular entry should provide compounds which overcome mechanisms of resistance to folate-based inhibitors of TS that are associated with decreased/altered folylpolyglutamate synthetase (FPGS) expression and/or an impaired RFC. Examination of a computer graphics model of the humanized Escherichia coli TS enzyme with quinazoline inhibitors of TS, such as 1 bound in the active site of the enzyme, suggested that conformational restriction introduced by bridging the C9 with C7 to form a pentacycle may be beneficial for binding to TS. That led to the synthesis of a series of potent cyclopenta[g]quinazoline-based inhibitors of the enzyme in which the glutamyl residue associated with classical antifolates was replaced with a variety of glutamate-derived ligands; the most potent inhibitor being the L-Glu-gamma-D-GluT(alpha) derivative 7j. In the mouse L1210:1565 cell line (mutant RFC), the majority of these compounds had activity equal or only slightly greater compared with the parental L1210 cell line, indicating a reduced dependence on the RFC for cellular uptake in the L1210 cell line.

Folate-based inhibitors of thymidylate synthase: synthesis and antitumor activity of gamma-linked sterically hindered dipeptide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583).

In an effort to synthesize inhibitors of thymidylate synthase (TS) that do not undergo polyglutamation, a series of gamma-linked sterically hindered dipeptide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) was prepared. A methyl, ethyl, or propargyl group was incorporated into the gamma-glutamyl amide bond of gamma-linked L,L dipeptide derivatives of ICI 198583, such as ICI 198583-gamma-L-Glu. In addition, steric bulk was introduced on either side of the gamma-glutamyl bond of ICI 198583-gamma-L-Glu or ICI 198583-gamma-L-Ala. The resulting dipeptide analogues, e.g., ICI 198583-gamma-MeGlu and ICI 198583-gamma-Aib, were apparently stable to in vivo hydrolysis but poorer inhibitors of TS and L1210 cell growth. However, introduction of 7-Me, 2'-F substitution into the quinazoline nucleus gave significant improvement in the inhibitory activity against thymidylate synthase. Compounds 28-30, the 7-Me, 2'-F derivatives of ICI 198583-gamma-MeGlu, ICI 198583-gamma-EtGlu, and ICI 198583-gamma-PgGlu, respectively, were potent inhibitors of TS (K(iapp) = 0.21-1.1 nM) and L1210 cell growth (IC50 = 0.05-0.34 microM) and were similar to that seen with the most potent gamma-linked L,D dipeptide derivatives of ICI 198583 previously synthesized. Furthermore, the low cross-resistance ratios for the L1210:R(D1694)/L1210 cell line indicated that 28-30 do not undergo polyglutamation.

Design and synthesis of potent non-polyglutamatable quinazoline antifolate thymidylate synthase inhibitors.

The synthesis is described of a series of analogues of the potent thymidylate synthase (TS) inhibitor, N-[4-[N-[(3,4-dihydro-2, 7-dimethyl-4-oxo-6-quinazolinyl)methyl]-N-prop-2-ynylamino]-2-f luorob enzoyl]-L-glutamic acid (4, ZM214888), in which the glutamic acid moiety is replaced by homologous amino acids and alpha-amino acids where the omega-carboxylate is replaced by acylsulfonamides and acidic heterocycles. In general these modifications when compared to 4 gave compounds with increased potency as inhibitors of isolated TS and as cytotoxic agents against murine tumor cell lines. The new compounds require transport by the reduced folate carrier for entry into cells but are not converted intracellularly into polyglutamated species. Agents with this profile are expected to show activity against tumors that are resistant to classical antifolates due to low expression of folylpolyglutamate synthetase. The analogue (S)-2-[4-[N-[(3,4-dihydro-2, 7-dimethyl-4-oxo-6-quinazolinyl)methyl]-N-prop-2-ynylamino]-2-f luorob enzamido]-4-(1H-1,2,3,4-tetrazol-5-yl)butyric acid (35, ZD9331) has been selected as a clinical development candidate and is currently undergoing phase I studies.

Quinazoline antifolates thymidylate synthase inhibitors: lipophilic analogues with modification to the C2-methyl substituent.

Modification of the potent thymidylate synthase (TS) inhibitor 1-[[N-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N- prop-2-ynylamino]benzoyl]amino]methyl]-3-nitrobenzene (4a) has led to the synthesis of quinazolinone antifolates bearing functionalized alkyl substituents at C2. A general synthetic route was developed which involved coupling the appropriate 1-[[N-[4-(alkylamino)benzoyl)amino]methyl]-3-nitrobenzene 20-22 with a 6-(bromomethyl)-2-(acetoxymethyl)-3,4-dihydro-4-oxoquinazoline 9 or 10. Replacement of the 2-acetoxy group by a chlorine atom followed by the displacement of the halogen of 25a-c by various nucleophiles led to compounds 26-40. Good TS (IC50 < 1 microM) and growth inhibition (IC50 0.1-1 microM) were found with most of these new antifolates. TS inhibitors in this series do not apparently require the reduced folate carrier (RFC) for cell entry (they most likely penetrate the cell membrane by passive diffusion) and are not polyglutamated. N, O, S, Cl, and CN as well as large amino and mercapto substituents were tolerated by the enzyme. The simultaneous incorporation of 7-methyl and 2'-F substituents gave a series of highly potent agents inhibiting cell growth at concentrations < 1 microM (24, 27bc; 30-32b, 35b). The incorporation of suitable C2 substituents has overcome the decrease in aqueous solubility observed with lipophilic quinazoline antifolates. This is best illustrated by compound 31a, where up to a 54-fold increase in solubility has been achieved by the incorporation of an N-methylpiperazine nucleus into the C2-methyl group of 4a.

New mustard prodrugs for antibody-directed enzyme prodrug therapy: alternatives to the amide link.

Antibody-directed enzyme prodrug therapy (ADEPT) is a two-step approach for the treatment of cancer which seeks to generate a potent cytotoxic agent selectively at a tumor site. In this work described the cytotoxic agent is generated by the action of an enzyme CPG2 on a relatively nontoxic prodrug. The prodrug 1 currently on clinical trial is a benzamide and is cleaved by CPG2 to a benzoic acid mustard drug 1a. We have synthesized a series of new prodrugs 3-8 where the benzamide link has been replaced by, for example, carbamate or ureido. Some of these alternative links have been shown to be good substrates for CPG2 and therefore new candidates for ADEPT. The active drugs 3a and 4a derived from the best of these prodrugs are potent cytotoxic agents (1-2 microM) some 100 times more than 1a. The prodrugs 3 and 4 are some 100-200-fold less cytotoxic, in a proliferating cell assay, than their corresponding active drugs 3a and 4a.

Resistance-modifying agents. 8. Inhibition of O(6)-alkylguanine-DNA alkyltransferase by O(6)-alkenyl-, O(6)-cycloalkenyl-, and O(6)-(2-oxoalkyl)guanines and potentiation of temozolomide cytotoxicity in vitro by O(6)-(1-cyclopentenylmethyl)guanine.

A series of O(6)-allyl- and O(6)-(2-oxoalkyl)guanines were synthesized and evaluated, in comparison with the corresponding O(6)-alkylguanines, as potential inhibitors of the DNA-repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT). Simple O(6)-alkyl- and O(6)-cycloalkylguanines were weak AGT inactivators compared with O(6)-allylguanine (IC(50) = 8.5 +/- 0.6 microM) with IC(50) values ranging from 100 to 1000 microM. The introduction of substituents at C-2 of the allyl group of O(6)-allylguanine reduced activity compared with the parent compound, while analogous compounds in the O(6)-(2-oxoalkyl)guanine series exhibited very poor activity (150-1000 microM). O(6)-Cycloalkenylguanines proved to be excellent AGT inactivators, with 1-cyclobutenylmethylguanine (IC(50) = 0.55 +/- 0.02 microM) and 1-cyclopentenylmethylguanine (IC(50) = 0.39 +/- 0.04 microM) exhibiting potency approaching that of the benchmark AGT inhibitor O(6)-benzylguanine (IC(50) = 0.18 +/- 0.02 microM). 1-Cyclopentenylmethylguanine also inactivated AGT in intact HT29 human colorectal carcinoma cells (IC(50) = 0.20 +/- 0.07 microM) and potentiated the cytotoxicity of the monomethylating antitumor agent Temozolomide by approximately 3- and 10-fold, respectively, in the HT29 and Colo205 tumor cell lines. The observation that four mutant AGT enzymes resistant to O(6)-benzylguanine also proved strongly cross-resistant to 1-cyclopentenylmethylguanine indicates that the O(6)-substituent of each compound makes similar binding interactions within the active site of AGT.

Quinazoline antifolate thymidylate synthase inhibitors: replacement of glutamic acid in the C2-methyl series.

The synthesis of a series of analogues of the potent thymidylate synthase (TS) inhibitor N-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6- quinazolinyl)methyl]-N-prop-2-ynylamino]benzoyl]-L-glutamic acid (ICI 198583, 1) is described in which the glutamic acid residue has been replaced by other alpha-amino acids. Most of these analogues were prepared by coupling of tert-butyl-4-(prop-2-ynylamino)benzoate (37) with 6-(bromomethyl)-3,4-dihydro-2-methyl-4-oxoquinazoline (34) followed by deprotection of the tert-butyl ester to the acid and azide-mediated coupling to the appropriate amino acid or amino acid ester. In cases where the amino acid ester was unreactive with the acid azide, a modification was used in which the quinazolinone moiety was protected as its 3-(pivaloyloxy)methyl derivative. This permitted the generation of the more reactive acid chloride of the p-aminobenzoate unit. In general these modifications result in compounds that have equivalent potency to 1 as inhibitors of isolated TS except where the amino acid lacks a lipophilic alpha-substituent. These compounds appear to require the reduced folate carrier (RFC) for transport into cells, but since they are not converted intracellularly into polyglutamated forms, they have a lower level of cytotoxicity compared to 1. The removal of the alpha-carboxylic acid has given a second set of analogues of 1 which contain simple alkyl amide, benzyl, substituted benzyl, and heterocyclic benzyl amide derivatives. These are considerably less potent than 1 as TS inhibitors but display 1-10 microM cytotoxicities due to the fact that they do not require RFC transport and can presumably readily enter cells by passive diffusion through the cell membrane. Molecular modeling and NMR studies indicated that the incorporation of, respectively, 7-methyl and 2'-fluoro substituents would favor the optimum conformation of these molecules for interaction with the TS enzyme. Accordingly, these substituents were incorporated into selected examples to give the series of analogues 47-55. These all show enhanced (approximately 10-fold) inhibition of TS compared to their unsubstituted counterparts. In the substituted benzylamides (51, 52) and heterocyclic benzyl amides (53-55) the ability to enter cells by passive diffusion results in highly potent (< 1 microM) cytotoxic agents.

Identification of novel purine and pyrimidine cyclin-dependent kinase inhibitors with distinct molecular interactions and tumor cell growth inhibition profiles.

Substituted guanines and pyrimidines were tested as inhibitors of cyclin B1/CDK1 and cyclin A3/CDK2 and soaked into crystals of monomeric CDK2. O6-Cyclohexylmethylguanine (NU2058) was a competitive inhibitor of CDK1 and CDK2 with respect to ATP (Ki values: CDK1, 5 +/- 1 microM; CDK2, 12 +/- 3 microM) and formed a triplet of hydrogen bonds (i.e., NH-9 to Glu 81, N-3 to Leu 83, and 2-NH2 to Leu 83). The triplet of hydrogen bonding and CDK inhibition was reproduced by 2,6-diamino-4-cyclohexylmethyloxy-5-nitrosopyrimidine (NU6027, Ki values: CDK1, 2.5 +/- 0.4 microM; CDK2, 1.3 +/- 0.2 microM). Against human tumor cells, NU2058 and NU6027 were growth inhibitory in vitro (mean GI50 values of 13 +/- 7 microM and 10 +/- 6 microM, respectively), with a pattern of sensitivity distinct from flavopiridol and olomoucine. These CDK inhibition and chemosensitivity data indicate that the distinct mode of binding of NU2058 and NU6027 has direct consequences for enzyme and cell growth inhibition.

The in vivo metabolic stability of dipeptide analogues of the quinazoline antifolate, ICI 198583, in mice.

In the search for quinazoline thymidylate synthase inhibitors that are not subject to intracellular polyglutamation, a class of dipeptide analogues of the diglutamate of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583-gamma-L-glu) has been evaluated for their stability to in vivo hydrolysis. Replacement of the second glutamate with another amino acid, e.g. alanine, prevented polyglutamation in vitro but such compounds were subject to hydrolysis when injected into mice. The extent of hydrolysis was measured in plasma, liver and kidney by HPLC analysis of tissue removed from mice 1 hr after i.p. injection. The enzyme responsible for this hydrolysis is thought to be a gamma-glutamyl hydrolase which hydrolyses the amide bond, releasing ICI 198583 which may then be polyglutamated. Development of stable dipeptide compounds was achieved by structural modification in two principal ways: either by replacement of the second amino acid (e.g. glutamate or alanine) with its D-enantiomer or removal of the carboxyl on the alpha-carbon of the second amino acid (alpha'-COOH). In this second approach two series of compounds were investigated. Monocarboxylate-derived dipeptides, e.g. ICI 198583-gamma-L-phenylalanine or ICI 198583-gamma-phenylglycine, resulted in stable compounds after removal of the alpha'-COOH (to give -ethylamide and -benzylamide derivatives, respectively). However, for the dicarboxylic amino acids a less clear picture emerged. Although removal of the alpha'-COOH from ICI198583-gamma-L-glutamate to give ICI 198583-gamma-gamma-aminobutyric acid resulted in a stable compound, the corresponding aspartate analogue (-beta-alanine) was subject to hydrolysis.

Synthesis and structure-activity relationships of a novel antifungal agent, ICI 195,739.

Antifungal azole derivatives are known to have potential for inhibition of host P-450 systems, and, in the attempts to increase the antifungal specificity of the inhibitor by identification of extra receptor binding within the enzyme complex, initial synthesis was guided by the structural requirements of the natural lanosterol substrate. With the aid of computer graphics, the 3'-styryl functionality was identified as a key structural element. For metabolically stable systems, in vitro-in vivo correlations exist, but optimizing oral activity resulted in the production of compounds with unacceptably long elimination half-lives. A disconnection of this relationship was achieved in pairs of structural isosteres with metabolic nonequivalence (CN:CONH2/OCH3:OCF3) and led to the identification of ICI 195,739, a novel 3'-tetrafluoropropoxystyryl-substituted bistriazole tertiary alcohol, as the compound of choice.

The role of the reduced-folate carrier and metabolism to intracellular polyglutamates for the activity of ICI D1694.

The uptake of ICI D1694 into L1210 cells is very rapid and evidence strongly suggests that transport is via the reduced-folate/MTX cell membrane carrier (RFC); for example a cell line with a greatly impaired RFC is highly resistant to ICI D1694. Polyglutamates can be found intracellularly within a few minutes, so that experiments initially designed to measure transport were actually measuring transport and polyglutamation. After 30mins, in normal serum-containing tissue culture medium, the concentration of polyglutamates (di, tri and tetra) exceeded that of the parent drug 6-fold. Studies where cells were resuspended in drug-free medium demonstrated that the parent drug and its diglutamate could readily leave the cell. Folinic acid could markedly decrease the polyglutamation of ICI D1694, but had to be given simultaneously with the drug as a 4hr delayed rescue was less effective because substantial polyglutamation had already occurred. This effect was translated into considerable antagonism for cell growth inhibition by simultaneous folinic acid. The importance of the metabolism of ICI D1694 to polyglutamates to its potent cytotoxic activity is demonstrated by compounds related in structure to ICI D1694 but with different properties for the RFC and FPGS. For example, 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolate (ICI 198583) owes its less potent cytotoxic activity to its poorer FPGS substrate activity (Km 40 microM compared with 1.3 microM for ICI D1694). Replacing the 2-methyl of either compound with amino, which appears to prevent use of the RFC, has a deleterious effect on growth inhibitory activity presumably by limiting the transport of the parent compounds into the cells, thereby slowing the rate of polyglutamate formation. Again a single change to another part of the molecule, that is methylation of the 7-position can have serious consequences on cytotoxic potency, particularly for the ICI D1694 molecule. The 7-methylated compounds are apparently poor or non-substrates for FPGS and therefore retain activity against a cell line unable to polyglutamate antifolates. These same compounds are only slightly affected by coincubation with folinic acid in L1210 tissue culture, consistent with the failure of these compounds to form intracellular polyglutamates. The results of short-exposure assays and in situ TS assays confirms that 7-methylation largely prevents the formation of a retained drug-form (polyglutamates), continuous exposure being necessary to maintain TS inhibition and cause a cytotoxic effect after removal of extracellular drug.(ABSTRACT TRUNCATED AT 400 WORDS)

Antifungal antibiotics from Calcarisporium thermophilum: a new source of 15-azahomosterol derivatives.

Two antifungal metabolites isolated from Calcarisporium thermophilum were identified as 15-azahomosterols related to the compounds previously isolated from Geotrichum flavo-brunneum. By full spectral comparison with authentic 15-aza-24-methylene-D-homocholesta-8,14-dien-3 beta-ol (A 25822 B) from G. flavo-brunneum, the Calcarisporium metabolites were characterized as the 4 alpha-methyl- and 4,4-dimethyl-analogues of A 25822 B. Several minor members of the series were also detected, and tentatively identified by MS analysis. The 15-azahomosterols exhibited good antifungal activity towards Candida parapsiliosis, though the activities were somewhat lower than that of the 4-demethyl derivative A25822 B. Calcarisporium thermophilum is the second microorganism known to synthesize these unusual 15-azahomosterol derivatives.