Synthesis and Discovery of Water Soluble Microtubule Targeting Agents that Bind to the Colchicine Site on Tubulin and Circumvent Pgp Mediated Resistance

Abstract

Two classes of molecules were designed and synthesized based on a 6-CH₃ cyclopenta[d]pyrimidine scaffold and a pyrrolo[2,3-d]pyrimidine scaffold. The pyrrolo[2,3-d]pyrimidines were synthesized by reacting ethyl 2-cyano-4,4-diethoxybutanate and acetamidine, which in turn was chlorinated and reacted with the appropriate anilines to afford 1 and 2. The cyclopenta[d]pyrimidineswere obtained from 3-methyladapic acid, followed by reaction with acetamidine to afford the cyclopenta[d]pyrimidine scaffold. Chlorination and reaction with appropriate anilines afforded (±)-3•HCl – (±)-7.HCl. Compounds 1 (add chemical compound designation) and (±)-3•HCl (add compound chemical designation) had potent antiproliferative activities in the nanomolar range. Compound (±)-3•HCl is significantly more potent than 1. Mechanistic studies showed that 1 and (±)-3•HCl cause loss of cellular microtubles, inhibit the polymerization of purified tubulin, and inhibit colchicine binding. Modeling studies show interactions of these compounds within the colchicine site. The identification of these new inhibitors that can also overcome clinically relevant mechanisms of drug resistence provides new scaffolds for colchicine site agents.

Introduction

Three major classes of microtubule active agents have been identified (Fig. 1).^1–3 The vinca alkaloids vincristine, vinblastine, vindesine and vinorelbine, are β-tubulin binding agents that are important in the treatment of leukemias, lymphomas, non-small cell lung cancer and other cancers. These are microtubule polymerization inhibitors. The second group include the taxoids, paclitaxel (Taxol) and docetaxel (Taxotere), as well as the epothilones. The binding site for paclitaxel is also in the β-subunit but is distinct from that of the vinca alkaloids. Paclitaxel and other taxoids (and the epothilones) bind to the interior of the microtubule,^4,5 and these are designated microtubule-stabilizing agents becuase they stimulate tubulin polymerization. The taxoids are useful in the treatment of breast, lung, ovarian, head and neck and prostate carcinomas among others. The third class, typified by colchicine, is comprised of a diverse collection of small molecules that bind to the colchicine site on β-tubulin at its interface with α-tubulin, distinct from the vinca site. Colchicine site agents also inhibit tubulin polymerization. Combretastatins, as exemplified by combretastatin A-4 (CA4) and its phosphorylated prodrug combretastatin A-4 phosphate (CA4P) are a class of drugs that are in clinical trials as antitumor agents that bind to the colchicine site.¹ Colchicine itself is not used as an antitumor agent but is used in the treatment of gout and familial Mediterranean fever. There are no clinically approved antitumor agents that bind to the colchicine site. However, several of these agents are currrently in clinical trials,^6,7 demonstrating the importance of developing colchicine site agents both for use alone and in combination chemotherapy protocols.

Figure 1.

Structures of microtubule targeting agents.

Half of all human tumors have mutations in the p53 gene, and the most effective drugs in p53 mutant cell lines are tubulin-binding agents.⁸ This further emphasizes the importance of developing new tubulin-binding drugs that are active against resistant tumors. Comprehensive reviews of structures and activities for these agents are available in the literature.^{9 – 13}

Despite the unprecedented success of microtubule disrupting agents in cancer chemotherapy, multidrug resistance (MDR) is a major limitation of cancer chemotherapy, and MDR tumors are usually resistant to tubulin-binding drugs. Overexpression of P-glycoprotein (Pgp) has been reported in the clinical setting in a number of tumor types, particularly after patients have received chemotherapy.^11,12 In addition, it has also been reported that Pgp expression may be a prognostic indicator in certain cancers and is associated with poor response to chemotherapy,^13,14 demonstrating the clinical importance of this mechanism of tumor resistance. Overexpression of Pgp appears to be more relevant in the clinical setting than elevation of MRP1 levels.¹⁵ The overwhelming lack of success of Pgp inhibitors in the clinic indicates that agents that are not susceptible to Pgp-mediated resistance is an important property for new microtubule targeting agents.^12,16 Such agents will fill an unmet need in the clinic for patients that develop resistance due to Pgp overexpression.

The other clinical mechanism of resistance to microtubule targeted agents involves the expression of specific isotypes of β-tubulin, of which the βIII-tubulin isotype is of paramount concern. There is unequivocal clinical evidence that βIII-tubulin expression is involved in resistance to taxoids and vinca alkaloids in multiple tumor types including non small cell lung,^17–19 breast,²⁰ and ovarian cancers.^21,22 Excellent reviews of the importance of βIII-tubulin in tumor resistance to microtubule targeted agents are available.²³ In a recent study, Stengel et al.²⁴ and Lee et al.²⁵ have shown that colchicine site agents circumvent βIII-tubulin resistance and highlight the critical importance of developing new agents that bind to the colchicine site as an alternative to the taxoids for the treatment of refractory cancers. The ability to overcome resistance due to Pgp and βIII-tubulin expression was part of the impetus for the recent FDA approval of ixabepilone as an antitumor agent and further attests to the importance of developing agents that overcome these resistance mechanisms.²⁶

An additional serious problem with several of the currently used antitubulin agents, particularly the taxoids, is their poor water solubility. This necessitates their formulation in polyoxyethylated castor oil or polysorbate, which can cause hypersensitivity reactions and require long administration times. This is a problem, too, with ixabepilone, which also needs to be dissolved in polyoxyethylated castor oil. Thus water soluble microtubule targeted agents are highly coveted, and an enormous effort continues to chemically modify and/or formulate analogs of these agents to increase their water solubility.

Synergistic or additive effects of microtubule targeted agents used in combination occurs, in part, due to the different or overlapping target sites of the agents on tubulin. Estramustine and paclitaxel, which bind to different sites on tubulin, can act synergistically or additively in combination.²⁷ Combinations including paclitaxel or docetaxel plus vinorelbine, are more effecatious in combination than as single agents.^{28, 29} CA4P is currently in clinical trials in combination with paclitaxel (www.clinicaltrials.gov).²⁹ As noted above, no colchicine site agent is FDA approved for cancer chemotherapy. There are however several colchicine site agents in clinical trials, and approval of such a drug would allow evaluation of whether enhanced activity could be obtained by combining it with a taxoid or vinca alkaloid. Thus new tubulin-binding agents that do not act at the taxoid or vinca alkaloid site on tubulin are of considerable interest, both as single agents and to combine with these established drugs.

Synthesis of water soluble microtubule targeting agents

We had previously synthesized 2-amino-4-anilino-6-substituted pyrrolo[2,3-d]pyrimidines as receptor tyrosine kinase (RTK) inhibitors of general structure A. (Fig. 2). Three possible binding modes to RTK were suggested from molecular modeling. To define the mode(s) of binding we synthesized the aniline N-methylated analog (A. R₁ = CH₃; R₂ = H), the N7-methylated analog (A. R₁=H; R₂ = CH₃) and the aniline N and N7-dimethylated analog (A. R₁ = CH₃; R₂ = CH₃). These results were first reported in 2005.^{30, 31} Further, analog design from these lead compounds afforded the pyrrolo[2,3-d]pyrimidines 1–2 and the cyclopenta[d]pyrimidine (±)-3 (Fig. 2) as the free base, and 1 – (±)-3 as the HCl, water soluble salts as well. Compounds 1 – (±)-3 were not RTK inhibitors (evaluated against EGFR, VEGFR-1 VEGFR-2 and PDGFR-β), but in the preclinical screening program of the National Cancer Institute in its 60 tumor cell line panel, 1 inhibited the proliferation of most of the 60 cancer cell lines with a GI₅₀ of less than 500 nM (Table 1) and (±)-3•HCl inhibited the proliferation of the majority of the the 60 cell lines with a potent GI₅₀ of less than 30 nM (Table 1).

Figure 2.

Lead compounds

Table 1.

Tumor cell growth inhibitory activity GI₅₀ (nM) of 1 and (±)-3•HCl in NCI 60 cell line panel.

Panel/ Cell line	GI50(nM) Compound		Panel/ Cell line	GI50(nM) Compound		Panel/ Cell line	GI50(nM) Compound		Panel/ Cell line	GI50(nM) Compound
Leukemia	1	(±)-3•HCI	Colon Cancer	1	(±)-3•HCI	Melanoma	1	(±)-3•HCI	Renal Cancer	1	(±)-3•HCI
CCRF-CEM	315	16.3	COLO 205	243	18.3	LOX IMVI	407	22.7	786 - 0	433	34.8
HL-60(TB)	190	<10	HCC-2998	271	24.0	M14	218	10.2	A498	198	<10
K-562	179	<10	HCT-116	344	15.1	MDA-MB-435	44.2	<10	ACHN	556	14.6
MOLT-4	503	31.8	HCT-15	275	<10	SK-MEL-2	251	33.1	CAKI-1	339
RPMI-8226		15.7	HT29	279	11.8	SK-MEL-28	603	<10	RXF 393	226	<10
SR	87.6	<10	KM12	181	<10	SK-MEL-5	177	<10	SN12C	854	31.8
NSCLC			SW-620	246	<10	UACC-62	625	<10	TK10	390	292
A549/ATCC	723	24.4	CNS Cancer			Ovarian cancer			UO-31	307	13.4
EKVX	875	19.6	SF-268	449	15.2	IGROVI	340	11.0	Prostate Cancer
HOP-62	291	19.4	SF-295	269	<10	OVCAR-3	192	<10	PC-3	509	14.6
HOP-92	4800	55.4	SF-539	288	11.4	OVCAR-4	7730	26.8	DU-145	269	21.4
NCI-H226	623	31.5	SNB-19	612	36.9	OVCAR-5	1670	38.5	Breast Cancer
NCI-H23	438	16.4	SNB-75	185	<10	OVCAR-8	639	32.8	MCF7	314	<10
NCI-H322M	428	59.0	U251	440	12.5	NCI/ADR-RES	164	<10	MDA-MB-231/ATCC	1260	24.6
NCI-H460	351	23.4				SK-OV-3	248	27.5	HS 578T	249	<10
NCI-H522	40.2	<10							BT-549		21.9
									MDA-MB-468	208	<10

The potent activities of these compounds prompted a COMPARE analysis,^{32, 34} which showed vincristine sulfate to have the closest Pearsons correlation coefficient with 1 and (±)-3•HCl. Other compounds, such as vinblastine sulfate and maytansine, also tubulin binding agents, were ranked as the next closest correlation. This clearly warranted the evaluation of 1 and (±)-3•HCl as tubulin binding agents. It was also of interest to synthesize and evaluate 2, the desmethyl analog of 1 and (±)-4, the desmethyl analog of (±)-3. In addition, due to the more potent activity of (±)-3•HCl in the NCI preclinical panel as compared to 1, compounds (±)-5•HCl– (±)-7•HCl (Fig. 2) were also synthesized to determine the position and importance of the 4′-OMe moiety to the biological activity of (±)-3•HCl.

Chemistry

Compound 1 was synthesized as shown in Scheme 1. Ethyl 2-cyano-4,4-diethoxybutanoate 8 and acetamidine•HCl, 9, were reacted at reflux for 5 h, cooled to 0 °C and the precipitate collected and treated with conc. H₂SO₄ in ethanol at reflux for 2 h to afford 2-methyl-4-oxo-pyrrolo[2,3-d]pyrimidine 10 in 64% yield. Chlorination with POCl₃ afforded 11 (83% yield).³⁵ Treatment of 11 with the appropriate anilines 12 afforded 1 (R = CH₃) (80% yield) and 2 (R = H) (41%). The water soluble hydrochloride salt of 1 was obtained as a white precipitate (~ 100%) by dissolving 1 in anhydrous ether followed by bubbling anhydrous HCl gas.

Scheme 1. Synthesis of pyrrolo[2,3-d]pyrimidines 1 and 2^a.

^aReagents and conditions: (a) i) 2-bromo-1,1-diethoxyethane, NaOMe, anhyd. DMF, 90 °C, 4 h. ii) acetamidine•HCl, NaOEt, EtOH, reflux, 3 h. iii) Conc. H₂SO₄, EtOH, reflux, 2 h. (b) POCl₃, reflux, 3 h. (c) i-PrOH, HCl (1 drop), reflux, 4 h. (d) anhyd. HCl gas, anhyd. ether.

Target compounds (±)-3 – (±)-7 were prepared as shown in Scheme 2. 2,6-Dimethyl-6,7-dihydro-3H-cyclopenta[d]pyrimidin-4(5H)-one (±)-14 was prepared via the reaction of commercially available 3-methyladipic acid (±)-13 and conc. H₂SO₄ at reflux in ethanol and a Dieckmann condensation in the presence of elemental sodium in toluene, followed by treatment with acetamidine•HCl 9 (21% over three steps). Chlorination of (±)-14 with POCl₃ for 3 h at reflux afforded (±)-15 (69%).³⁶ Compound (±)-15 was unstable and easily decomposed to (±)-14 if not used immediately. Reaction of (±)-15 with the appropriate anilines in i-PrOH in the presence of 2–3 drops of conc. HCl afforded (±)-3 – (±)-7. Anhydrous HCl gas was bubbled through an anhydrous ether solution of (±)-3, (±)-5 – (±)-7 to afford the corresponding HCl salts as white solids.

Scheme 2. Synthesis of cyclopenta[d]pyrimidines (±)-3 – (±)-7 and their HCl salts^a.

^aReagents and conditions: (a) i) conc. H₂SO₄, EtOH, reflux, 8 h. ii) Na, toluene, reflux, 3 h. iii) acetamidine• HCl 9, t-BuOK, t-BuOH. (b) POCl₃, reflux, 3 h. (c) i-PrOH, HCl (2–3 drops), reflux, 3–6 h. (d) anhyd. HCl gas, anhyd. ether.

Biological evaluations

Compounds 1 and (±)-3•HCl showed potent GI₅₀s in most of the NCI 60 cancer cell lines (Table 1). The microtubule disrupting effects of 1 and (±)-3•HCl were observed (Fig. 3) in a cell-based phenotypic screen. Compounds 1 and (±)-3•HCl caused dramatic reorganization of the interphase microtubule network, similar to the effects of colchicine and CA4P. The EC₅₀ (concentration required to cause 50% loss of cellular microtubules) (Table 2) was calculated to be 7 nM for CA4P²⁵, 56 nM for (±)-3•HCl and 5.8 μM for 1. Consistent with effects on cellular microtubules, 1 and (±)-3•HCl caused the formation of aberrant mitotic spindles and mitotic accumulation when measured by flow cytometry (Fig.4 and S3). Compounds 1 and (±)-3•HCl are potent microtubule depolymerizers in cells, confirming the COMPARE analysis results.

Figure 3. The effects of CA4P, 1 and (±)-3•HCl on interphase microtubules.

A-10 cells were treated with vehicle or compounds as indicated for 18 h, and then the cells were fixed and microtubules visualized by indirect immunofluorescence techniques.

Table 2.

IC₅₀ values for inhibition of proliferation of MDA-MB-435 cells and EC₅₀s for cellular microtubule loss.

Cmpd	IC50±SD (MDA-MB-435)	EC50 for microtubule depolymerization
1	183 ± 3.4nM	5.8 μM
2	>10 μM	> 40μM
(±)-3•HCl	17.0 ± 0.7 nM	56 nM
(±)-4	>10 μM	> 40 μM
(±)-5•HCl	153 ± 11.1 nM	3.0 μM
(±)-6•HCl	ND	> 40 μM
(±)-7•HCl	2.7 ± 0.3 μM	>40 μM

Figure 4.

Cell cycle distribution.

MDA-MD-435 cells were treated with vehicle (A), 12.5 nM paclitaxel (B), 45 nM (±)-3•HCl (C), or 850 nM 1 (D) for 24 h. The cells were then harvested, stained with propidium iodide and evaluated by flow cytometry.

Cell cycle effects of 1 and (±)-3•HCl

Antiproliferative effects

Compounds 1 and (±)-3•HCl were tested for antiproliferative effects against the drug sensitive MDA-MB-435 cell line using the sulforhodamine B assay (SRB assay).^{37, 38} The data (Table 2) indicate that (±)-3•HCl has potent antiproliferative effects with an IC₅₀ (concentration required to cause 50% inhibition of proliferation) of 17.0 ± 0.7 nM. Compound 1 is less potent, consistent with its effects in the phenotypic assay and by flow cytometry, with an IC₅₀ of 183 ± 3.4 nM.

The ability of 1 and (±)-3•HCl to circumvent Pgp-mediated drug resistance was evaluated by using an SK-OV-3 isogenic cell line pair (Table 3). In this cell line pair the Rr of paclitaxel, a well known Pgp substrate is greater than 800 while Rr values of 1.2 and 1.6 were obtained with 1 and (±)-3•HCl respectively, consistent with the Rr values obtained with other colchicine site agents, CA4P and 2ME2 of 1.5–2.6. These data suggest that both 1 and (±)-3•HCl are poor substrates for transport by Pgp and thus have advantages over some clinically useful tubulin-targeting drugs like paclitaxel.

Table 3.

1 and (±)-3•HCl circumvent Pgp and βIII-tubulin mediated resistance.

Drug	Effect of Pgp on drug sensitivity*			Effect of βIII-tubulin on drug sensitivity**
	IC50 (SD) (nM)		Rr	IC50 (SD) (nM)		Rr
	SK-OV-3	SK-OV-3 MDR-1-6/6		HeLa	WT βIII
Paclitaxel	3.0 ± 0.06	2,600 ± 270	864	1.6 ± 0.2	7.7 ± 0.2	4.7
CA4P	4.5 ± 0.2	6.6 ± 1.3	1.5	4.7 ± 0.2	5.7 ± 0.4	1.2
2ME2	867 ± 36	2,268 ± 235	2.6	608 ± 55	586 ± 37	1
1	278 ± 19	435 ± 33	1.6	270 ± 21	186 ± 17	0.7
(±)-3•HCI	38.6 ± 3.1	44.4 ± 3.2	1.2	37.3 ± 4.1	23.9 ± 1.7	0.6

Key: Rr – Relative resistance.

^*Antiproliferative effects of (±)-3•HCl and 1 in parental and MDR-1-transducted cell lines in comparison with other microtubule disrupting agents. The IC₅₀ values were determined using the SRB assay. (n=3 ± SD). The Rr was calculated by dividing the IC₅₀ of the Pgp overexpressing cell line by the IC₅₀ of the parental cell line.

^**Effects of the expression of βIII-tubulin on the sensitivity of cell lines to microtubule-targeting agents. The Rr was calculated by dividing the IC₅₀ of the WT βIII cell line by the IC₅₀ of the parental HeLa cells.

A second mechanism of drug resistance that can lead to treatment failure with tubulin-targeting drugs is the expression of the βIII isotype of tubulin as discussed above. An isogenic HeLa cell line pair was used to evaluate the effect of βIII tubulin expression on the activities of 1 and (±)-3•HCl.³⁹ Compounds 1 and (±)-3•HCl have Rr values of 0.6–0.7 (Table 3) in this cell line pair suggesting that they overcome drug resistance mediated by βIII tubulin as compared with paclitaxel, which has a Rr of 4.7. Thus both 1 and (±)-3•HCl inhibit tumor cells with equal potency without regard to their expression of Pgp or βIII-tubulin.

Further mechanistic studies

Studies were conducted to determine if 1 and (±)-3•HCl inhibited the polymerization of purified bovine brain tubulin, as would be predicted from the effects in cells. These biochemical studies provide an indication of a direct interaction of the compounds with tubulin. An initial study shown in Fig. 5, indicate that both 1 and (±)-3•HCl are potent inhibitors of purified tubulin assembly. Both compounds were therefore compared with CA4P as inhibitors of assembly in a quantitative study (Table 4). In this assay, both 1 and (±)-3•HCl inhibited tubulin assembly about as well as CA4P (Table 4). The data in Table 4 also shows that 1 and (±)-3•HCl bind at the colchicine site on tubulin, since they inhibited [³H]colchicine binding to the protein, although not as potently as CA4P.

Figure 5. Effects of microtubule disrupting agents on tubulin polymerization.

Paclitaxel represents a positive control for a microtubule stabilizer and colchicine the control for a microtubule depolymerizer. The assays were conducted using purified bovine brain tubulin from Cytoskeleton Inc in the presence of 10% glycerol, 3 mg/mL purified tubulin and 10 mM GTP.

Table 4.

Inhibition of tubulin assembly and inhibition of colchicine binding.

Cmpd.	Inhibition of tubulin assembly	Inhibition of colchicine binding
	IC50 (μM) ± SD	% Inhibition ± SD
		1 μM	5 μM
CA4P	1.0 ± 0.09	88 ± 2	99 ± 0.2
1	2.6±0.05	ND	70±2
(±)-3•HCl	1.9 ± 0.01	60 ± 2	84 ± 3

To determine the structural requirements for the 4′-OCH₃ and N4-CH₃ moieties for activity, compounds 2 and (±)-4-(±)-7 were synthesized. The superior activity of (±)-3•HCl suggests that the pyrrole NH of 1 is not important for activity. Table 2 indicates that the methylation of the aniline N is crucial for activity in both scaffolds (compare 1 with 2 and (±)-3•HCl with (±)-4). In addition, the 4′-OCH₃ moiety is important for potent activity (compare (±)-3•HCl with (±)-7•HCl), and the optimum location for the methoxyl group is at the 4′-position since (±)-5•HCl, which has a 3′-OCH₃, was about 9-fold less potent than (±)-3•HCl, which has a 4′-OCH₃. Finally, (±)-6•HCl, the analog with a 2′-OCH₃, was inactive.

Molecular modeling

In an attempt to explain the molecular basis of the potent activity of the N-Me analogs 1 and (±)-3•HCl and the inactivity of the N-desmethyl analogs 2 and (±)-4, we modeled compounds 1 – 7 into the colchicine site. The X-ray crystal structure of tubulin at 3.58 Å resolution was obtained from the protein database (PDB ID 1SA0⁴⁰). This crystal structure contains the αβ dimers of tubulin complexed with N-deacetyl-N-(2-mercaptoacetyl)colchicine (DAMA colchicine), a close structural analog of colchicine. The binding of colchicine to the tubulin dimer has been described in the literature.^40–42 Colchicine binds to β tubulin at its interface with α tubulin. The colchicine site has dimensions of ~10 Å × ~10 Å × ~4–5 Å and is composed of strands S8 and S9, loop T7 and helices H7 and H8 (nomenclature and amino acid residue positions as described in ref. 39) from the β subunit and loop T5 from the α subunit of tubulin. Thrα179 and Valα181 in the α-tubulin subunit form hydrogen bonds with colchicine. Additionally, Cysβ241 forms a hydrogen bond with the oxygen atom of the 3- OCH₃ in the A-ring of colchicine. Further hydrophobic stabilization is afforded by side chain atoms of Valα181 and Metβ259. In addition, the carbonyl group of the A-ring H-bonds with Lysβ352.^{41, 42}

Docking of (R)-3

Docking studies were performed using the docking suite of Molecular Operating Environment software (MOE 2008.10).⁴³ Details of the docking protocol used are provided in the experimental section. Multiple low-energy conformations (within 1kcal/mol of the best pose) were obtained on docking (R)-3 and other analogs. The multiple docked poses can be explained by the large volume of the active site (10 Å × 10 Å × 4–5 Å).⁴¹ Figure 6 shows the docked conformation of (R)-3 which was arbitrarily selected as a working model for the docking of compounds 1 – 7 on the basis of their structural similarity to the bound conformation of DAMA colchicine. The pose in Fig. 6 for (R)-3 had a score (−6.8379 kcal/mol) within 1 kcal/mol of the best scored pose. Comparison of the docked conformation of (R)-3 with the crystal structure conformation of DAMA colchicine shows overlap of the 4′-OCH₃ phenyl group of (R)-3 with the trimethoxybenzene A-ring of DAMA colchicine (Fig. 6). In this pose the 4′-OCH₃ of (R)-3 overlaps with the 3′-OCH₃ group in the A ring of DAMA colchicine. Similar interactions of the -OCH₃ groups with Cysβ241 have been reported in the literature.^{41, 44} The conformation of (R)-3 depicted in Fig. 6 permits the formation of a hydrogen bond between Cysβ241 and the oxygen atom of the 4′- OCH₃ of (R)-3 as is observed between Cysβ241 and the 3′-OCH₃ group of DAMA colchicine in its X-ray crystal structure with tubulin. The phenyl ring of (R)-3 mimics the A ring of colchicine and is involved in hydrophobic interactions with amino acids from β-tubulin (Leuβ248, Alaβ250, Leuβ255, and Alaβ316). Additionally, the methyl group of the 4′-OCH₃ moiety could interact with the side chain of Ileβ378 and/or with the side chain of Valβ318.

Figure 6.

Stereo view. Docked pose of (R)-3 (white) overlaid with DAMA colchicine (red) in the colchicine site of tubulin.

We^45–49 and others⁵⁰ have previously demonstrated that similar CH₃ moieties dictate the biological activities predicated on hydrophobic interactions and/or conformational restriction. It was of interest to extend these findings to provide a rationale for the high potency of the N-CH₃ analogs of the present study compared with the N-desmethyl analogs.

The N- CH₃ group of (R)-3 occupies a region in space in proximity to the C5 and C6 atoms of the B-ring of DAMA colchicine. In this orientation the N-CH₃ group is involved in hydrophobic interactions with the side chain C atoms of Lysβ254 and Alaβ250. An additional hydrophobic interaction between the N- CH₃ moiety of (R)-3 and the side chain C-atom of Leuβ248 also occurs due to the flexible nature of the protein (measured distance between N-CH₃ and side chain C of Leuβ248 is 4.21Å). These interactions could assist in stabilization of the docked conformation of (R)-3 and could explain, in part, the observed difference in activity of the N-desmethyl analog, (R)-4, which would lack these additional interactions (Fig. 7). The N-CH₃ also aids in maintaining the relative conformations of the cyclopenta[d]pyrimidine and the phenyl ring. While similar docked poses were observed for (R)-4, the docked poses of compounds with the N-CH₃ group consistently scored higher (~1 kcal/mol) than those of compounds that lack the N-CH₃ group.

Figure 7.

Stereo view. Superimposition of the docked poses of (R)-3 (white) and (R)-4 (magenta) in the colchicine site of tubulin.

A systematic conformational search carried out using Sybyl × 1.1⁵¹ for energy minimized structures of (R)-3 and (R)-4 returned a lower number of conformers for the N-CH₃ compound (R)-3 compared with the NH compound (R)-4 using the same settings. This is indicative of restrictions on the rotational freedom of the 4′-OCH₃ phenyl ring of (R)-3 in the presence of the N-CH₃ moiety and decreases the number of low energy conformations of the 4′-OCH₃ phenyl ring relative to the cyclopenta[d]pyrimidine scaffold of (R)-3. Similar results were obtained for the conformational search of 1 and 2.

The cyclopenta[d]pyrimidine of (R)-3 ring partially overlaps with the C-ring of DAMA colchicine and is stabilized by hydrophobic interactions with side chain C atoms of Leuβ255, Asnβ258 and Lysβ252. The C7 of (R)-3 overlaps the C9 carbonyl C of DAMA colchicine. The C2-CH₃ moiety of (R)-3 is involved in a hydrophobic interaction with Alaα180 (4.35Å) while the C6-CH₃ group of (R)-3 is involved in hydrophobic interactions with Valα181 and Alaβ316. We did not observe a significant difference in the binding poses and docked scores of (R)-3 (−6.8379 kcal/mol) and (S)-3 (−6.9454 kcal/mol) due to the chirality of the C6-CH₃ group (Fig. 8). Whether this slight predicted difference between the two isomers is reflected in the biological activities can only be determined once the two isomers are individually synthesized and evaluated.

Figure 8.

Stereo view. Superimposition of the docked poses of (R)-3 (white), (S)-3 (cyan) and DAMA colchicine (red) in the colchicine site of tubulin.

While this work was in progress, studies describing a series of quinazolines^51–54 and thieno[3,2-d]pyrimidines⁵⁵ as potent apoptosis inducers were published. These reports suggest a similar function for the N-CH₃ moiety, but do not provide molecular details about the binding modes of the quinazolines and/or thieno[3,2-d]pyrimidines to tubulin.

Molecular modeling suggests that the binding interactions afforded by the 4′-OCH₃ group on the anilino ring plays an important part and dictates the potency of these compounds against tubulin, since deletion or moving the 4′-OCH₃ moiety results in a significant loss of activity. However, molecular modeling does not provide a reason(s) for the loss of potency of (±)-6 (2′-OCH₃) compared with (±)-3. We speculate that the loss of potency could be explained either by the loss of interactions of the 4′-OCH₃ moiety of (±)-3 and/or the additional conformational restriction of the anilino ring by a combination of N-methylation and the 2′-OCH₃ moiety on the phenyl ring or both. The docking mode of 1 (not shown) was strikingly similar to the docking mode of (R)-3. The pyrrole NH of 1 was not involved in a hydrogen bond with the amino acid residues of the tubulin binding site. Additionally, 1 lacks the 6-CH₃ substitution in (R)-3 and could thus lack the additional bonding afforded by the substituent at C6. The docked pose of 1 had a score of −6.0330 kcal/mol, somewhat less but comparable to (R)- and (S)-3.

We also compared the binding mode of compounds 1 – 7 to the proposed binding modes⁴¹ of 15 known tubulin inhibitors. As an example, (R)-3 retains the key binding interactions exhibited by the known tubulin inhibitors and is in a conformation consistent with those for the reported compounds⁴¹ (Fig. 9). Similar docked poses were observed in this study for compounds 1 – 7.

Figure 9.

Stereo view. Docking mode of (R)-3 (white) overlaid with docked poses of 15 ligands⁴¹ (blue) in the colchicine site of tubulin.

In summary, we report the synthesis and identification of the mechanism of action of water soluble, colchicine site microtubule depolymerizing agents 1 and (±)-3•HCl. These compounds inhibit the growth of tumor cells with GI₅₀ values in the nanomolar range and overcome two clinically important tumor resistance mechanisms that limit activity of microtubule targeting agents, expression of Pgp and βIII tubulin. For these analogs the 6-CH₃ cyclopenta[d]pyrimidine scaffold is better than the pyrrolo[2,3-d]pyrimidine scaffold, and both the 4-N-methyl and 4′-OCH₃ groups on the aniline ring are required for optimal potency. We have also provided an explanation of the importance of the N-CH₃ moiety of 1 and (±)-3•HCl for biological activity via molecular modeling. Compounds 1 and (±)-3•HCl are important lead analogs for the development of further compounds for in vivo evaluation and possible advancement into clinical trials.

Experimental

Analytical samples were dried in vacuo (0.2 mmHg) in a CHEM-DRY drying apparatus over P₂O₅ at 50 °C. Melting points were determined on a digital MEL-TEMP II melting point apparatus with FLUKE 51 K/J electronic thermometer and are uncorrected. Nuclear magnetic resonance spectra for proton (¹H NMR) were recorded on Bruker Avance II 400 (400 MHz) and 500 (500 MHz) NMR systems. The chemical shift values are expressed in ppm (parts per million) relative to tetramethylsilane as an internal standard: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad singlet. Thin-layer chromatography (TLC) was performed on Whatman Sil G/UV254 silica gel plates with a fluorescent indicator, and the spots were visualized under 254 and 366 nm illumination. Proportions of solvents used for TLC are by volume. Column chromatography was performed on a 230 – 400 mesh silica gel (Fisher Scientific) column. Elemental analyses were performed by Atlantic Microlab, Inc., Norcross, GA. Element compositions are within ±0.4% of the calculated values and indicates > 95% purity. Fractional moles of water or organic solvents frequently found in some analytical samples could not be prevented despite 24 – 48 h of drying in vacuo and were confirmed where possible by their presence in the 1H NMR spectra. Mass spectrum data was acquired on the Agilent G6220AA TOF LC/MS system using the nano ESI (Agilent chip tube system with infusion chip). All solvents and chemicals were purchased from Sigma-Aldrich Co. or Fisher Scientific Inc. and were used as received.

2-Methyl-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (10)

The synthesis of 10 utilized a reported method.³⁵ To solution of ethyl 2-cyanoacetate 8 (10 mmol, 1.13 g) in anhydrous DMF (20 mL) was added sodium methoxide (10 mmol, 0.54 g). After stirring for 30 min, 2-bromo-1,1-diethoxyethane (10 mmol, 1.97 g) was added, and the reaction was heated at 90 °C for 4 h. After cooling to room temperature, the reaction solution was extracted with diethyl ether (2×20 mL). The ether layer was collected, dried over sodium sulfate and evaporated to give a pale yellow liquid. The liquid was added to a solution of acetamidine hydrochloride 9 (10 mmol, 0.94 g) and sodium ethoxide (10 mmol, 0.68 g) in ethanol (20 mL) and refluxed for 3 h. The solution was evaporated to dryness and extracted with ethyl acetate and water. The organic layer was collected and evaporated to afford a solid. Concentrated sulfuric acid (2 mL) in ethanol (10 mL) was added to the solid, and the mixture was kept at reflux for 2 h. At the end of the reaction, water (10 mL) was added and the pH was adjusted to 8 using ammonium hydroxide. The precipitate obtained was collected and dried to give 10 as an off-white powder (0.85 g). Compound 10 was used without further purification.

4-Chloro-2-methyl-7H-pyrrolo[2,3-d]pyrimidine (11)

Compound 10 (5 mmol, 0.75 g) was treated with POCl₃ (10 mL) and heated under reflux for 3 h and the remaining POCl₃ was removed in vacuo. Ammonium hydroxide was carefully added to the resulting slurry, followed by extraction with ethyl acetate. After combining the organic phases and evaporation, compound 11 was obtained as a pale yellow powder (0.67 g, 81%) and used without further purification. R_f 0.45 (CH₃OH:CHCl₃ = 1:5). An analytical sample was purified for ¹H NMR (DMSO-d₆): δ 2.28 (s, 3 H, 2-CH3), 6.35 (d, 1 H, 5-H, J = 3.28 Hz), 6.93 (d, 1 H, 6-H, J = 3.28 Hz), 11.67 (s, 1 H, 7H, D₂O exchanged).

N-(4-Methoxyphenyl)-N,2-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (1)

To a solution of compound 11 (5 mmol, 0.84 g) in isopropanol (4 mL) was added 4-methoxy-N-methylaniline 12 (5.5 mmol, 0.75 g) and a drop of conc. HCl. The reaction mixture was refluxed for 4 h. After evaporation of the solvents, a silica gel plug (2 g) was made and loaded to a silica gel column. The column was eluted sequentially with 0% and 1% methanol in chloroform. Fractions containing the product (TLC) were pooled and evaporated to give afford 1 as a white solid (1.14 g, 85%). R_f 0.60 (CH₃OH:CHCl₃ = 1:5); mp, 229.3 – 229.5 °C. ¹H NMR (DMSO-d₆): δ 2.45 (s, 3 H, 2-CH₃), 3.45 (s, 3 H, NCH₃), 3.82 (s, 3 H, OCH₃), 4.52 (d, 1 H, 5-H, J = 3.50Hz), 6.73 (d, 1 H, 6-H, J = 3.55 Hz), 7.04 (d, 2 H, phenyl, J = 8.85 Hz), 7.27 (d, 2 H, phenyl, J = 8.85 Hz), 11.27 (s, 1 H, 7-H, D₂O exchanged). HRMS m/z calcd for C₁₅H₁₇N₄O [M+H]⁺, 269.1397; found, 269.1385 [M+H]⁺. Anal. (C₁₅H₁₆N₄O) C, H, N.

N-(4-Methoxyphenyl)-N,2-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine•HCl (1·HCl)

The HCl salt of 1 was obtained by treating its ether/ethyl acetate solution with HCl gas in an ice bath for 5 min. The white salt as it formed precipitated from the solution. The salt was collected and dried to give a white solid, mp 247.5 – 249.7 °C. ¹H NMR (DMSO-d₆): δ 2.64 (s, 3 H, 2-CH₃), 3.62 (s, 3 H, NCH₃), 3.81 (s, 3 H, OCH₃), 4.59 (s, 1 H, 5-H), 7.00 (s, 1 H, 6-H), 7.12 (d, 2 H, phenyl, J = 8.45 Hz), 7.41 (d, 2 H, phenyl, J = 8.30), 12.32 (s, 1 H, 7-H, D₂O exchanged). Anal. (C₁₅H₁₆N₄O) C, H, N.

N-(4-Methoxyphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (2)

The synthesis of 2 followed the same method as for 1 using 11 (5 mmol, 0.84 g) and 4-methoxy- aniline 12 (5.5 mmol, 0.68 g). After column chromatography with 0% and 1% methanol in chloroform, fractions containing the product (TLC) were pooled and evaporated to afford 2 as a white solid (0.95 g, 75%). R_f 0.42 (CH₃ OH:CHCl₃ = 1:5); mp, 258.7 − 259.9 °C. ¹H NMR (DMSO-d₆): δ 2.42 (s, 3 H, 2-CH₃), 3.73 (s, 3 H, OCH₃), 6.55 (d, 1 H, 5-H, J = 3.24 Hz), 6.91 (d, 2 H, phenyl, J = 8.96 Hz), 7.05 (d, 1 H, 6-H, J = 3.44 Hz), 7.74 (d, 2 H, phenyl, J = 8.92 Hz), 9.02 (s, 1H, N-H), 11.41 (s, 1 H, 7-H, D₂O exchanged). HRMS m/z calcd for C₁₄H₁₅N₄O [M+H]⁺, 255.1240; found, 255.1234 [M+H]⁺. Anal. (C₁₄H₁₄N₄O) C, H, N.

2,6-Dimethyl-6,7-dihydro-3H-cyclopenta[d]pyrimidin-4(5H)-one ((±)-14)

3-Methyladipic acid (±)-13 (1.60 g, 10 mmol) was heated at reflux in ethanol/conc. sulfuric acid solution (35 mL, v/v = 2.5/1) for 8 h. The solution was neutralized with ammonium hydroxide to pH = 7, then diluted with ethyl acetate (100 mL) and washed with water. The organic phase was dried with anhydrous sodium sulfate and evaporated to afford a light yellow liquid which was used in the next step without further purification. The yellow liquid was dissolved in anhydrous toluene (100 mL) and sodium (0.23 g) was added to the solution. The mixture was heated at reflux for 3 h and cooled, neutralized with 1N hydrochloric acid solution and washed with water. After drying with anhydrous sodium sulfate, the organic phase was separated and evaporated to afford a light brown liquid. The liquid was used in the next step without further purification. The light brown liquid was diluted with t-BuOH. Acetamidine hydrochloride (1.13 g, 12 mmol) and potassium tert-butoxide (1.34 g, 12 mmol) were added, and the mixture was heated at reflux for overnight. The reaction mixture was cooled and the precipitate was collected by filtration. The residue was washed with warm methanol twice (30 mL ×1, 15 mL ×1). The filtrate and washings were combined, and then 3 g silica gel was added and removed the solvent in vacuo to afford a dry plug. This plug was placed on the top of a silica gel column and eluted with 1% methanol in chloroform. Fractions containing the product were pooled and evaporated to afford 0.35 g of (±)-14 (21% yield total for 3 steps) as a white solid: TLC R_f 0.30 (CHCl₃/CH₃OH, 10:1); mp 173.9–175.4 °C; ¹H NMR (DMSO-d₆) δ 1.05–1.07 (d, 3 H, CH₃, J = 6.8 Hz), 2.24 (s, 3 H, CH₃), 2.13–2.34, 2.73–2.90 (m, 5 H, CH₂CHCH₂), 12.16 (br, 1 H, OH, exch); Anal. (C₉H₁₂N₂O) C, H, N.

4-Chloro-2,6-dimethyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine ((±)-15)

A mixture of (±)-14 (0.25 g, 1.5 mmol) and POCl₃ (10 mL) was heated at reflux for 3 h. The reaction mixture was cooled and evaporated at reduced pressure. The residue was diluted with chloroform (50 mL) and neutralized with ammonium hydroxide slowly in an ice bath. The organic portion was washed with water (3 × 30 mL) and dried with anhydrous sodium sulfate. Concentration of the organic solvent with 1 g silica gel afforded a dry plug. This plug was placed on the top of a silica gel column and eluted with 20% hexane in chloroform. Fractions containing the product were pooled and evaporated to afford 0.14 g of (±)-15 as a colorless liquid (69%). Compound (±)-15 was unstable and was used for the next step without further characterization.

General procedure for (±)-3 -(±)-7

Compound (±)-15 and aniline or the appropriate substituted N-methyl anilines were dissolved in isopropanol (5 mL). To this solution was added 37% Hydrochloric acid (2–3 drops). The mixture was heated at reflux for 3–6 h. Then the reaction was cooled and evaporated at reduced pressure. The residue was diluted with chloroform, neutralized with ammonium hydroxide, and then washed with water (2 × 30 mL). After drying with anhydrous sodium sulfate, organic solvent with 1 g silica gel was evaporated under reduced pressure to give a dry plug. This plug was placed on the top of a silica gel column and eluted with chloroform. Fractions containing the product were pooled and evaporated to afford pure compounds (±)-3 - (±)-7. Compounds were dissolved in anhydrous ether (10 mL), and then anhydrous hydrochloric acid gas was bubbled into the solution till no more solid precipitated out. The white solid was collected by filtration and dried over P₂O₅ to afford (±)-3•HCl, (±)-5•HCl-(±)-7•HCl.

N-(4-Methoxyphenyl)-N,2,6-trimethyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-aminium chloride ((±)-3•HCl)

Compound (±)-3 was synthesized from (±)-15 (0.19 g, 1.01 mmol) and 4-methoxy-N-methyl aniline (0.17 mg, 1.22 mmol) using the general procedure described above to afford after purification 0.18 g (61%) as a light yellow liquid: TLC R_f 0.24 (CHCl₃/CH₃OH, 10:1). Compound (±)-3•HCl was synthesized from (±)-3 as a white solid (0.16 g, 79%): mp 196–198 °C; 1H NMR (DMSO-d₆) δ 0.85–0.86 (d, 3 H, CH₃, J = 6.8 Hz), 1.37–1.47 (m, 1 H, CH), 1.89–1.99 (m, 1 H, CH), 2.26–2.35 (m, 1 H, CH), 2.43–2.44 (m, 1 H, CH), 2.98–3.05 (m, 1 H, CH), 2.60 (s, 3 H, 2-CH₃), 3.51 (s, 3 H, NCH₃), 3.80 (s, 3 H, OCH₃), 7.01–7.03 (d, 2 H, Ph-H, J = 8.8 Hz), 7.33–7.35 (d, 2 H, Ph-H, J = 8.8 Hz), 14.88 (br, 1 H, HCl, exch). Anal. (C₁₇H₂₂N₃OCl) C, H, N, Cl.

N-(4-Methoxyphenyl)-2,6-dimethyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-amine ((±)-4)

Compound (±)-4 was synthesized from (±)-15 (0.17 g, 0.94 mmol), 4-methoxyaniline (0.14 g, 1.13 mmol) using the general procedure described above to afford after purification 0.16 g (61%) as a white solid: TLC R_f 0.32 (CHCl₃/CH₃OH, 10:1); mp 130.5–131.3 °C; H NMR (DMSO-d₆): δ 1.09–1.12 (d, 3 H, CH₃, J = 6.8 Hz), 2.35 (s, 3 H, 2-CH₃), 1.36–1.48 (m, 1 H, CH), 1.88–2.01 (m, 1 H, CH), 2.25–2.35 (m, 1 H, CH), 2.41–2.45 (m, 1 H, CH), 2.98–3.05 ((m, 1 H, CH)), 3.73 (s, 3 H, OCH₃), 6.86–6.89 (d, 2 H, Ph-H, J = 8.8 Hz), 7.60–7.63 ((d, 2 H, Ph-H, J = 8.8 Hz)), 8.41 (br, 1 H, NH, exch). Anal. (C₁₆H₁₉N₃O) C, H, N.

N-(3-Methoxyphenyl)-N,2,6-trimethyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-aminium chloride ((±)-5•HCl)

Compound (±)-5 was synthesized from (±)-15 (0.23 g, 1.3 mmol) and 3-methoxy-N-methyl aniline (0.20 g, 1.4 mmol) using the general procedure described above to afford after purification as a light yellow liquid: TLC R_f 0.33 (CHCl₃/CH₃OH, 10:1). Compound (±)-5•HCl was synthesized from (±)-5 as a off white solid (0.27 g, 66% for two steps): mp 209.6–210.7 °C; ¹H NMR (DMSO-d₆) δ 0.85–0.87 (d, 3 H, CH₃, J = 6.8 Hz), 1.44–1.50 (m, 1 H, CH), 1.95–2.01 (m, 1 H, CH), 2.26–2.35 (m, 1 H, CH), 2.45–2.48 (m, 1 H, CH), 3.00–3.07 (m, 1 H, CH), 2.62 (s, 3 H, 2-CH₃), 3.55 (s, 3 H, NCH₃), 3.78 (s, 3 H, OCH₃), 6.69–7.42 (m, 4 H, Ph-H), 15.21 (br, 1 H, HCl, exch). Anal. (C₁₇H₂₂N₃OCl• 0.1H₂O) C, H, N, Cl.

N-(2-Methoxyphenyl)-N,2,6-trimethyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-aminium chloride ((±)-6•HCl)

Compound (±)-6 was synthesized from (±)-15 (0.24 g, 1.31 mmol) and 2-methoxy-N-methyl aniline (0.18 g, 1.31 mmol) using the general procedure described above to afford after purification as a colorless liquid: R_f 0.37 (CHCl₃/CH₃OH, 10:1). Compound (±)-6•HCl was synthesized from (±)-6 as a white solid (0.23 g, 55% for two steps): mp 148–149 °C; ¹H NMR (DMSO-d₆) δ 0.83–0.85 (d, 3 H, CH₃, J = 6.8 Hz), 1.31–1.39 (m, 1 H, CH), 1.82–1.93 (m, 1 H, CH), 2.26–2.31 (m, 1 H, CH), 2.43–2.46 (m, 1 H, CH), 2.99–3.07 (m, 1 H, CH), 2.62 (s, 3 H, 2-CH₃), 3.45 (s, 3 H, NCH₃), 3.79 (s, 3 H, OCH₃), 7.03–7.51 (m, 4 H, Ph-H), 15.09 (br, 1 H, HCl, exch). Anal. (C₁₇H₂₂N₃OCl•0.1H₂O) C, H, N, Cl.

N-Phenyl-N,2,6-trimethyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-aminium chloride ((±)-7•HCl)

Compound (±)-7 was synthesized from (±)-15 (0.18 g, 0.99 mmol) and N-methyl aniline (0.11 g, 0.99 mmol) using the general procedure described above to afford after purification 0.21 g (83%) as a white solid: R_f 0.36 (CHCl₃/CH₃OH, 10:1); mp 112.6–113.2 °C; ¹H NMR (DMSO-d₆) δ 0.81–0.83 (d, 3 H, CH₃), 1.40–1.44, 1.86–2.26, 2.74–2.80 (m, 5 H, CH₂CHCH₂), 2.41 (s, 3 H, 2-CH₃), 3.37 (s, 3 H, NCH₃), 7.18–7.41 (m, 4 H, Ph-H). Anal. (C₁₆H₁₉N₃) C, H, N. Compound (±)-7•HCl was synthesized from (±)-7 (0.17 g, 0.65 mmol) as a white solid (0.15 g, 79%): mp 263.2–264.6 °C; ¹H NMR (DMSO-d₆) δ 0.83–0.85 (d, 3 H, CH₃, J = 6.8 Hz), 1.35–1.42 (m, 1 H, CH), 1.81–1.91 (m, 1 H, CH), 2.24–2.32 (m, 1 H, CH), 2.42–2.45 (m, 1 H, CH), 2.99–3.05 (m, 1 H, CH), 2.61 (s, 3 H, 2-CH₃), 3.55 (s, 3 H, NCH₃), 7.41–7.53 (m, 4 H, Ph-H), 15.09 (br, 1 H, HCl, exch). Anal. (C₁₆H₂₀ClN₃) C, H, N, Cl.

Molecular Modeling and Computational Studies

The X-ray crystal structure of tubulin at 3.58 Å resolution was obtained from the protein database (PDB ID 1SA0⁴⁰). This crystal structure contains the αβ dimers of tubulin complexed with N-deacetyl-N-(2-mercaptoacetyl) colchicine (DAMA colchicine), a close structural analog of colchicine.

Docking Procedure

Docking studies were performed using the docking suite of Molecular Operating Environment software (MOE 2008.10).⁴³ The PDB file was downloaded (www.pdb.org) and imported into MOE. The A and B subunits of the protein, along with the crystallized ligand, were retained while the C, D, and E subunits, GTP, GDP and Mg ions were deleted. After addition of hydrogen atoms, the protein was then “prepared” using the LigX function in MOE. LigX is a graphical interface and collection of procedures for conducting interactive ligand modification and energy minimization in the active site of a flexible receptor. In LigX calculations, the receptor atoms far from the ligand are held fixed (constrained not to move) while receptor atoms near the ligand (in the active site) are allowed to move but are subject to tether restraints that discourage gross movement. The procedure was performed with the default settings.

Ligands were built using the molecule builder function in MOE and were energy minimized to its local minima using the MMF94X forcefield to a constant (0.05 kcal/mol). Ligands were docked into the active site of the prepared protein using the docking suite as implemented in MOE. The docking was restricted to the active site pocket residues using the Alpha triangle placement method. Refinement of the docked poses was carried out using the Forcefield refinement scheme and scored using the Affinity dG scoring system. Around 30 poses were returned for each compound at the end of each docking run. The docked poses were manually examined in the binding pocket to ensure quality of docking and to confirm absence of steric clashes with the amino acid residues of the binding pocket.

To validate the utility of MOE 2008.10 for docking ligands into the active site, DAMA colchicine, the native ligand in the crystal structure (PDB: 1SA0) was built using the molecule builder, energy minimized and docked into the active site using the above parameters. The best docked pose of DAMA colchicine displayed an RMSD of 0.9531 Å compared with the crystal structure pose of DAMA colchicine. MOE 2008.10 was thus validated for our docking studies. Docking studies of the lead compounds and selected proposed molecules was performed using the same procedure. Coordinates of the binding models of the 15 known tubulin inhibitors reported by Nguyen et al.⁴¹ was retrieved from the supporting information section accessible online. The coordinates as well as the docked pose of (R)-3 were imported and visualized using MOE 2008.10. A systematic conformational search was carried out using Sybyl × 1.1⁵¹ using 5° increments. Molecules were built using the molecule builder function in MOE 2008.10 and were imported into Sybyl × 1.1 and energy minimized using the Tripos forcefield with a distance-dependent dielecetric and the Powell conjugate gradient with a convergence criterion of 0.01 kcal/mol.

Cellular studies

Effects of compounds on cellular microtubules

A-10 cells were used to evaluate the effects of the compounds on interphase and mitotic microtubules using indirect immunofluorescence techniques and the EC₅₀ values were calculated form a minimum of 3 experiments as described previously.²⁵

Sulforhodamine B (SRB) Assay

The SRB assay^{37, 38} was used to evaluate the antiproliferative and cytotoxic effects of the compounds against cancer cells as previously described.³⁹ MDA-MB-435, SK-OV-3 and HeLa cells were purchased from the American Type Culture Collection (Manassas, VA). The methods use to generate and characterize the SK-OV-3 MDR-1-6/6 and WTβIII cell lines have been described.³⁹ The IC₅₀ represents the mean of at least 3 independent experiments using triplicate points in each experiment.

In vitro tubulin polymerization

Inhibition of tubulin polymerization was measured using the tubulin polymerization assay kit (Cytoskeleton Inc.) according to manufacturer's directions. Briefly, 300 μg of purified bovine brain tubulin was incubated with tubulin assembly buffer (80 mM Na-Pipes, pH 6.9, 1 mM EGTA, 1 mM MgCl₂, 10 mM GTP and 10% glycerol) and the indicated drug in a final volume of 100 μl. The polymerization of purified tubulin into microtubules was monitored by absorbance at 340 nm at 37°C in a Spectromax Plus 96 well plate spectrophotometer (Molecular Devices).

Cell cycle analysis

MDA-MB-435 cells were treated for 24 h with vehicle, 1 or (±)-3•HCl or 12.5 nM paclitaxel as a positive control. Following drug treatment, the cells were harvested and stained with Krishan's reagent and analyzed using a FacsCalibur flow cytometer. The data are plotted as propidium iodide intensity versus the number of events.

Quantitative tubulin studies

Bovine brain tubulin was purified as described previously.⁵⁷ The assembly reaction mixtures contained 1.0 mg/mL (10 μM) tubulin, 0.8 M monosodium glutamate (pH of 2 M stock solution adjusted to pH 6.6 with HCl), 4% (v/v) dimethyl sulfoxide as compound solvent, varying compound concentrations, and, after a 15 min preincubation at 30 °C, 0.4 mM GTP. The 0 °C reaction mixtures were transferred to 0 °C cuvettes in a recording spectrophotometer equipped with an electronic temperature controller. After baselines were established, the temperature was jumped to 30 °C over about 30 s, and changes in turbidity were measured over 20 min. The compound concentration that caused a 50% reduction in increase in turbidity, interpolated from the values obtained with defined compound concentrations, was defined as the IC₅₀ value.⁵⁸ The assay to measure inhibition of [³H]colchicine binding was described in detail previously.⁵⁹ Reaction mixtures contained 0.1 mg/mL (1.0 μM) tubulin, 5.0 μM [³H]colchicine, and potential inhibitors at 1.0 or 5.0 μM, as indicated. Incubation was for 10 min at 37 °C, at which point the reaction has reached 40–60% of the maximum colchicine that can be bound in reaction mixtures without inhibitor. The [³H]colchicine was a product of Perkin-Elmer. CA4P was a generous gift of Dr. G. R. Pettit, Arizona State University.

Abbreviations

(CA4)

Combretastatin A-4

(MDR)

multiple drug resistance

(Pgp)

P-glycoprotein

(MRP1)

Multidrug resistance protein 1

(RTK)

receptor tyrosine kinase

(EGFR)

epidermal growth factor receptor

(PDGFR)

platelet-derived growth factor receptor

(VEGFR)

vascular endothelial growth factor receptor

(Rr)

Relative resistance

(DAMA colchicine)

N-deacetyl-N-(2-mercaptoacetyl)colchicine

(2ME2)

2-methoxyestradiol