Ortho Group Activation of a Bromopyrrole Ester in Suzuki-Miyaura Cross-Coupling Reactions: Application to the Synthesis of New Microtubule Depolymerizing Agents with Potent Cytotoxic Activities

Abstract

New microtubule depolymerizing agents with potent cytotoxic activities have been prepared with a 5-cyano or 5-oximino group attached to a pyrrole core. The utilization of ortho activation of a bromopyrrole ester to facilitate successful Suzuki-Miyaura cross-coupling reactions was a key aspect of the synthetic methodology. This strategy allows for control of regiochemistry with the attachment of four completely different groups at the 2, 3, 4 and 5 positions of the pyrrole scaffold. Biological evaluations and molecular modeling studies are reported for these examples.

1. Introduction

The efficient and regiocontrolled preparation¹ of highly functionalized pyrroles continues to be a very active area of research as the result of biologically important pyrrole² containing natural products and their synthetic counterparts. Suzuki-Miyaura cross-coupling reactions offer an excellent synthetic opportunity to functionalize brominated pyrroles regiochemically but such reactions are known to be problematic when the pyrrole nitrogen is unsubstituted as demonstrated by Handy and coworkers³ (Scheme 1). In this example reduction of the organometallic intermediate becomes a significant side reaction.

Scheme 1.

Suzuki-Miyaura Cross-Coupling studies of 4-bromo-2-carbethoxypyrrole

Handy and coworkers have also shown³ that when the nitrogen is substituted with various nitrogen protecting groups, much better yields (70–80% range) are obtained for these cross-coupling transformations. Buchwald and co-workers⁴ have reported the successful use of two new precatalysts for such cross-coupling reactions as applied to various nitrogen unsubstituted heterocycles (indoles and pyrazoles). However, no examples of N-unsubstituted pyrroles were presented.

We recently reported⁵ a partial solution to this problem by the use of a 5-formyl group to activate such 4-bromo-2-carbethoxypyrroles (Scheme 1, 1) for successful Suzuki-Miyaura cross-coupling reactions. Additionally, our research groups have a long term interest⁶ in exploring highly functionalized pyrroles as colchicine site microtubule depolymerizers and we now report the application of this ortho activation methodology to the synthesis of some new and uniquely functionalized, unsymmetrical pyrroles, which exhibit significant cytotoxic actions.

The biological activities of compounds 4a and 4b (Fig. 1) were recently reported.⁷ Compound 4a was particularly interesting, since it displayed potent activity as a colchicine site microtubule depolymerizer with low nanomolar antiproliferative and cytotoxic activities against multiple cancer cell lines. In contrast, compound 4b was much less potent as both a microtubule depolymerizer and cytotoxin.⁷ This comparison suggests that introducing smaller substituents than bromine at the 3-and 5-positions could weaken the overall fit of these molecules within the colchicine binding site on tubulin.

Fig. 1.

Examples of two bioactive, synthetic pyrroles

However, there has been some concern in the literature⁸ regarding the toxicity of certain dibrominated pyrroles related to the discovery of the drug atorvastatin. The dibromo-pyrrole, PD 123244-15, caused severe toxicities in rats related to induction of liver HMG-CoA reductase and localized esophageal and forestomach irritations when administered by oral gavage. These toxicities were associated with high plasma drug concentrations.⁸ Compound 4a is also quite hydrophobic and for these reasons appropriate modifications of compound 4a seemed reasonable to pursue. In addition, we previously established that the 4-(2,3,4-trimethoxyphenyl) group and the 2-carbethoxy group of the pyrrole scaffold^6,9 are optimum for the inhibition of microtubule formation and cancer cell proliferation. Consequently, we have focused our attention on the introduction of selective functional group changes at the 5-position of compound 4a in anticipation of minimizing potential toxicity issues while maximizing pharmacodynamic and pharmacokinetic properties. Our initial choice for substituent replacements at the 5-position were small cyano or oximino groups given their compact size, lower molecular weight relative to bromine, greater polarity and the additional SAR insights, which would be provided by their preparation and bioassay. We now report both the synthetic and biological studies to this end.

2. Results and Discussions

The synthesis of 4-bromo-5-cyano-2-carbethoxypyrrole (7, Scheme 2) was accomplished by first converting our previously reported formylpyrrole building block⁵ (5, Scheme 2) to a mixture of oxime isomers (6, Scheme 2). This was followed by treatment of the crude oxime mixture with phosphorous oxychloride at room temperature in chloroform, producing the desired cyanopyrrole (7, Scheme 2) in an overall 71% yield from the formylpyrrole building block.

Scheme 2.

Preparation of 4-bromo-5-cyano-2-carbethoxypyrrole building block

As part of our broad based synthetic studies we also wanted to determine the ability of the cyano group to activate an ortho bromine on our pyrrole scaffold in the Suzuki-Miyaura cross-coupling reaction as it relates to the inability of non-activated, non-nitrogen protected systems to effectively accomplish this end. With the desired starting material (7) in hand, we examined a variety of aryl and heteroaryl boronic acids under our standard cross-coupling conditions⁵ and these results are described in Scheme 3 and Table 1.

Scheme 3.

Suzuki-Miyaura cross-coupling reactions of ethyl 3-bromo-2-cyanopyrrole-5-carboxylate with various aryl and heteroarylboronic acid derivatives

Table 1.

Reactions of various aryl and heteroaryl boronic acid derivatives with ethyl 3-bromo-2-cyanopyrrole-5-carboxylate under Suzuki-Miyaura cross-coupling conditions.

Entry	Compound	Ar	% Yield Isolated
1	8a	4-MeOPh	91
2	8b	3,4-(MeO)2Ph	100
3	8c	3,4,5-(MeO)3Ph	80
4	8d	4-CF3OPh	62
5	8e	3-MeOPh	81
6	8f	4-MePh	53
7	8g	4-ClPh	82
8	8h	3,4-(Cl)2Ph	67
9	8i	Ph	66
10	8j	4-(CO2Me)Ph	63
11	8k	4-(COMe)Ph	87
12	8l	4-Furan-3-yl	75
13	8m	4-Thien-3-yl	96
14	8n	2,3,4-(MeO)3Ph	70

The isolated yields for the various cross-coupled pyrroles were quite good irrespective of the electron releasing or electron withdrawing properties of the substituents attached to the aromatic group of the boronic acid. Heterocyclic groups such as furan and thiophene were also efficiently accommodated by our standard cross-coupling conditions. In comparison to the earlier studies regarding formyl group activation of the 4-bromo-2-carbethoxypyrrole (Scheme 5, 5) towards Suzuki-Miyaura cross-coupling reactions, the cyano analog (Scheme 3, 7) gave very similar results. Compound 8n in Table 1 was the required precursor to obtain the 5-cyano analog of NT-7-16 (Fig. 1, 4a) and this transformation was accomplished by treatment of 8n with KOH and NBS in DMF at room temperature (Scheme 4), in which case an 87% yield of the desired product (9) was obtained. It should also be noted that this sequence, which leads to KL-3-95 (9), provides a very unique tetrasubstitued pyrrole with four completely different substituents and with complete control of regiochemical specificity.

Scheme 5.

Preparation of the 5-position oxime analog of NT-7-16

Fig. 4.

Computational model of NT-7-16, KL-3-95, NT-7-45 (Z) and NT-7-45 (E) in the colchicine Binding site of β-tubulin. Inset: close-up of substitutions at the 5-position of pyrrole.

Note: beige – NT-7-16; cyan – KL-3-95; orange – NT-7-45 (E); magenta – NT-7-45 (Z)

Scheme 4.

Preparation of 5-cyano position analog of NT-7-16

The preparation of the oxime analog (NT-7-45, 12) of NT-7-16 (4a) was accomplished by a slightly different sequence as illustrated in Scheme 5. The 5-formyl pyrrole building block (5) utilized in Scheme 2 was crossed-coupled with 2,3,4-trimethoxyphenyl boronic acid under our standard ortho activation conditions⁵ to yield a 2,4,5-trisubstituted pyrrole (10) in good yield (75%), which was then treated with KOH, NBS in DMF at room temperature. The resulting 3-bromo analog (11, 96% yield) was then reacted with hydroxylamine in pyridine with heating and the desired oxime analog NT-7-45 (12) was obtained as a mixture of Z (12a) and E (12b) isomers in good overall yield (68%). The stereoisomeric mixture was separated by flash chromatography into the pure Z (12a) and pure E (12b) isomers.

As was indicative for the preparation of the 5-cyano analog (9) of NT-7-16, the oxime analogs (12a and 12b, Scheme 5) represent 2,3,4,5-tetrasubstitued pyrroles with four different groups attached and were prepared with regiochemical specificity.

The biological evaluations of KL-3-95 (9) and the NT-7-45 isomers (12a and 12b) were conducted using standard protocols^7,10,11 and are presented in Table 2. These results show that KL-3-95 (9), NT-7-45 (Z) (12a) and NT-7-45 (E) (12b) exhibit low nanomolar potency in a panel of cancer cell lines including MDA-MB-435 melanoma cells, HeLa cervical carcinoma cells and SK-OV-3 ovarian cancer cells. The ability of 9, 12a and 12b to overcome clinically relevant drug resistance mechanisms including the expression of the βIII isotype of tubulin or the expression of the ABC transporter, P-glycoprotein (Pgp) were evaluated using isogenic pairs of cell lines. HeLa WT-βIII cells express βIII tubulin and expression of this tubulin isotype had only modest effects on sensitivity of the cells to KL-3-95, NT-7-45 (Z) or NT-7-45 (E) as indicated by relative resistance values of 1.3, 1.1 and 1.4 respectively. The efficacy of these three compounds was additionally evaluated in the Pgp-expressing multidrug resistant SK-OV-3 MDR-1-/M-6-6 cells and relative resistance values of 1.6, 1.5 and 1.8, respectively, were obtained demonstrating that these compounds circumvent Pgp-mediated drug resistance.

Table 2.

Antiproliferative potency of NT-7-16 analogs in a panel of cancer cell lines and their microtubule depolymerizing activity in comparison to other pyrroles IC₅₀ ± SD (nM)

Compound	MDA-MB-435a (nM)	HeLaa (nM)	HeLa WT βIIIa (nM)	Rr	SK-OV-3a (nM)	SK-OV-3 MDR-1/M-6-6a (nM)	Rr	EC50a (nM)	EC50/IC50	HINT Score
NT-7-16c (4a)	10.4 ± 0.5	12.9 ± 0.8	15.7 ± 0.2	1.2	15.2 ± 0.8	10.6 ± 0.3	0.70	37	3.6	824
NT-9-21c (4b)	116 ± 4	131 ± 8	140 ± 20	1.1	210 ± 70	140 ± 8 0	0.67	1000	8.6	772
KL-3-95 (9)	19.6 ± 1.8	20.6 ± 1.9	26.1 ± 0.8	1.3	26.2 ± 2.1	42.2 ± 5.5	1.6	88	4.5	588
NT-7-45 (Z) (12a)	25.3 ± 3.1	29.4 ± 1.0	31.3 ± 0.5	1.1	33.7 ± 1.3	49.6 ± 2.4	1.5	108	4.2	554
NT-7-45 (E) (12b)	14.0 ± 0.8	18.0 ± 0.9	25.0 ± 1.4	1.4	23.1 ± 0.5	41.6 ± 1.9	1.8	56	4	687
NT-7-45 (53:47 mix of E and Z) (12)	2.7 ± 0.2	5.0 ± 0.3	4.1 ± 1.0	0.9	5.3 ± 0.2	6.6 ± 1.2	1.2	9.2	3.4	ND
Paclitaxel	2.0 ± 0.2	1.5 ± 0.1	21.1 ± 1.8	14	3.0 ± 0.3	2330 ± 150	776	ND	ND	ND

^aIC₅₀ values are the mean of at least 3 independent experiments each conducted in triplicate ± SD.

^bn=3

^cFrom reference 7.

The effects of the compounds on cellular microtubules were visualized in A-10 cells using indirect immunofluorescence techniques as previously described.⁷ Microtubules (green) and nuclei (blue) in vehicle-treated cells are shown in Figure 2, panel A. The effects of a 15 nM concentration of the NT-7-45 isomer mixture (12) on cellular microtubules is shown in panel B where extensive loss of microtubules can be visualized. A wide range of concentrations of each compound were evaluated and the percentage of cellular microtubules that were depolymerized was estimated visually.⁷ The EC₅₀ values, the concentration that causes 50% loss of cellular microtubules was determined by linear regression. EC₅₀ values are presented in Table 2 and the EC₅₀/IC₅₀ ratio is indicative of the relationship between microtubule depolymerization and antiproliferative potency.

Figure 2.

Effects of NT-7-45 isomeric mixture on cellular microtubules

These results show that KL-3-95 (9), NT-7-45 (Z) (12a) and NT-7-45(E) (12b) have activities similar to the lead compound NT-7-16 (4a) but slightly lower potency and are far superior to the effects of NT-9-21 (4b). Interestingly, the bioassay results and the structure-based modeling-derived HINT scores (vide infra) for NT-7-45 (Z) (12a) and NT-7-45(E) (12b) indicate more potent microtubule depolymerizing and cancer cell inhibitory effects for the E isomer as compared to the Z isomer. It is of significant interest to note that the mix of the two isomers (NT-7-45,12) when subjected to the same panel of biological screens as the other pyrroles exhibited consistently lower IC₅₀’s and EC₅₀’s (Table 2) than either of the individual oxime isomers (12a and 12b). The reason for the higher potency of the isomeric mix of oximes versus the pure Z and E isomers is unclear at this point.

The IC₅₀ values were calculated from dose response curves generated using the sulforhodamine B (SRB) assay¹⁰ and the values represent the mean ± SD of at least 3 independent experiments. Relative resistance (R_r) values were calculated for the isogenic cell line pairs by dividing the IC₅₀ of the expressing cell line by the IC₅₀ of the parental cells.

Because of its superior in vitro potency as compared to NT-7-16, we evaluated the antitumor activity of the NT-7-45 mixture of isomers (12) in the MDA-MB-435 xenograft model of cancer.⁷ Mice were treated with NT-7-45 (12) i.p. daily at 75 mg/kg for a total of 9 days. Paclitaxel, 20 mg/mg, was used as a positive control and was administered i.p. on days 0, 2, 4, 6, 8, and 11. The average tumor volumes ± SEM for the 10 tumors in each treatment group are shown in Figure 3A. NT-7-45 caused statistically significant (p = 0.0005) inhibition of tumor growth on day 14 as did paclitaxel in this model (p < 0.0001). Statistical significance was determined for the treatments as compared to control using a 2-way ANOVA followed by Dunnett’s post-hoc test to correct for multiple comparisons. No overt toxicities were noted with NT-7-45, but paclitaxel at this dose and schedule caused modest average weight loss of 2.5 % on day 14. At the end of the trial, on day 14, the tumors were removed and weighed and the results, Figure 3B, show that the NT-7-45 isomer mixture (12) caused a statistically significant reduction in mean tumor mass on day 14 as compared to the control (p = 0.04) as determined using an unpaired, two-tailed Student’s t-test.

Figure 3.

NT-7-45 inhibits tumor growth in a MDA-MB-435 xenograft model

*** p = 0.0005, ***** p < 0.0001, * p = 0.04.

We have continued to study⁹ with modeling, the nature of the binding of compounds such as NT-7-16 (4a) within the colchicine site of microtubules and an analogous computational examination of KL-3-95 (9) and NT-7-45 Z (12a) and E (12b), respectively follows. Our previous work suggested that the ethyl ester substitution^6a at the 2-position of the pyrrole ring and the 2,3,4-trimethoxy substitution^6b,7,9 at the 4-position are ideal for microtubule depolymerization and antiproliferative activity in these compounds. Substitutions at the 3-position appear to be spatially constrained by the pocket, which is hydrophobic in the corresponding region. Our recent efforts have focused on 5-position pyrrole substitutions that appear to offer opportunities for improving both the binding and physiochemical properties of analogues, as our models suggest that this position is at the entrance of the pocket. Thus, the new synthetic methodology reported here yielding KL-3-95 (9) and NT-7-45 (12a and 12b) now becomes very important to our long-range goals.

KL-3-95 (9), NT-7-45 (12a and 12b) and NT-7-16 (4a) were docked in the colchicine site of tubulin using our previously reported modeling tools and procedures.^6,7,9,12 The modeled KL-3-95 (9), NT-7-45 (12a and 12b) and NT-7-16 (4a) complexes are shown in Figure 4. NT-9-21 is perfectly superposed on NT-7-16, and thus not shown. Post-docking scoring and evaluation were performed with HINT,¹⁴ which has proven to be useful in our previous studies of colchicine site microtubule destabilizers. The overall HINT score (Table 2) often correlates with measured binding and/or activity; similarly, the significance of individual functional groups, protein residues and/or interactions amongst a collection of compounds can be quantitated.⁶ While both the –Cl and –Br at the pyrrole 5-position in NT-9-21 and NT-7-16, respectively, make hydrophobic interactions with Leu248 and Ala250 the HINT score does recognize that –Cl is a poorer substituent than –Br (NT-9-21 vs. NT-7-16), but fails to indicate the magnitude of the difference perhaps due to entropic issues not otherwise accounted for, e.g., the smaller –Cls have fit into their subpockets somewhat looser than –Brs. Another possible contributing factor might be related to the small differences in polar associative capabilities between –Cl and –Br. The cyano compound, KL-3-95, appears to be placing the polar nitrile nitrogen in that mostly hydrophobic environment, as are both oxime isomers of NT-7-45. However, the E isomer seems preferred over the Z isomer in both the HINT scores and measured IC₅₀s, possibly because the E isomer’s OH group may be able to interact with backbone atoms on the surrounding hydrophobic residues. We also may need to consider the possibility of water-mediated interactions in this very solvent accessible part of the pocket. When a larger set of 5-substituted analogs are eventually synthesized, tested and modeled, we should be able to understand these data more completely.⁹

3. Conclusions

We have described new examples for the ortho activation of a 4-bromo-2-carbethoxypyrrole (7) in the Suzuki-Miyaura cross-coupling reaction. This synthetic strategy allows for control of regiochemistry in providing new, completely unsymmetrical 2,3,4,5-tetrasubstituted pyrroles. The resulting compounds KL-3-95 (9), NT-7-45 (Z) (12a), NT-7-45(E) (12b) and the isomeric mix (NT-7-45, 12) of oximes are the first examples of these bioactive pyrroles with much smaller polar substituents at the 5-position that retain much of the biological efficacy displayed by the brominated lead (NT-7-16, 4a). The bioassay studies of KL-3-95 (9), NT-7-45 (Z) (12a), NT-7-45(E) (12b) and NT-7-45 (12) indicate that they have low nanomolar potency for microtubule depolymerization and cytotoxic activities against cancer cell lines, including those that are multidrug resistant. Such information also provides significant insight into SAR-guided studies of new and more potent inhibitors of cancer cell proliferation. Future studies might also evaluate the effects of these compounds on endothelial cells. The initial microtuble active pyrrole of this series, JG-03-14, has multiple effects on endothelial cells that suggest potential antivascular activities.¹³

It remains unknown why the isomeric 12a/12b mix is more potent than either of the isolated components, but a few of the possible explanations are intriguing: 1) solvent molecules play an unexpectedly large but sensitive role in managing the binding site, particularly around the pyrrole 5-substituents at the pocket entrance; 2) the oxime Z isomer may engage in an intramolecular hydrogen bond, which impacts pharmacodynamics; 3) the mixture may self-associate in such a way as to affect cell penetration or other physiochemical properties; 4) the slight difference noted in the R_r values of the isomers for the HeLa cell line pair suggests the possibility that they interact slightly differently among tubulin isotypes, thus leading to superadditive effects when the mixture is used in cells that contain multiple isotypes. This may be more than an academic question because the answer might reveal a strategy for activity enhancement in this or other similar series of compounds.

4. Experimental

4.1 General

All chemicals were used as received from the manufacturer (Aldrich Chemicals and Fisher Scientific). All solvents were dried over 4 angstrom molecular sieves prior to their use. NMR spectra were obtained on either a Bruker 300 MHz spectrometer, or a Bruker 500 MHz spectrometer in either CDCl₃, d₆-DMSO or d₆-acetone solutions. IR spectra were recorded on a Nicolet Avatar 320 FT-IR spectrometer with an HATR attachment. High resolution mass spectra were obtained on a Shimadzu IT-TOF mass spectrometer at the University of Richmond. Low resolution GC-MS spectra were obtained on a Shimadzu QP 5050 instrument. Melting points and boiling points are uncorrected. Chromatographic purifications were carried out on a Biotage SP-1 instrument or a Biotage Isolera instrument (both equipped with a silica cartridge). Gradient elution with ethyl acetate/hexane was accomplished in both instances. The reaction products were normally eluted within the range of 4–8 column volumes of eluant with a gradient mixture of 60:40 ethyl acetate:hexane. TLC analyses were conducted on silica plates with hexane/ethyl acetate as the eluant. All purified reaction products gave TLC results, flash chromatograms, and ¹³C NMR spectra consistent with a sample purity of >95%.

4.1.1 4-Bromo-5-cyano-1H-pyrrole-2-carboxylic acid ethyl ester (7)

Into a 100 mL round bottom flask equipped with magnetic stirring and a reflux condenser was placed 4-bromo-5-formyl-1H-pyrrole-2-carboxylic acid ethyl ester (2.03 mmol), hydroxylamine hydrochloride (2.03 mmol), pyridine (0.2 mL) and ethanol (20 mL). The mixture was heated at reflux for two hrs, cooled to room temperature and concentrated in vacuo. The crude residue was partitioned between water (15 mL) and ethyl acetate (10 mL) and after phase separation the aqueous phase was extracted two additional times with 10 mL portions of ethyl acetate. The combined organic phases were washed with brine (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to yield 0.519 g of a brown solid (98% yield of oxime as a mixture of E and Z isomers). This crude material was then placed in a 100 mL round bottom flask equipped with a magnetic stirring bar and addition funnel. To the crude residue (1.92 mmol) was added anhydrous chloroform (20 mL) and the solution was stirred in an ice bath for 5 mins. Phosphorous oxychloride (1.92 mmol) in 10 mL of anhydrous chloroform was placed in the addition funnel and added dropwise with stirring to the crude oxime over a 10 min period. The ice bath was removed and the reaction mixture was stirred at room temperature overnight. The reaction mixture was subsequently quenched with water (30 mL) and the phases were separated followed by washing the organic phase with brine (30 mL) and dried over anhydrous sodium sulfate. The drying agent was removed by filtration and the solvent was removed in vacuo to give 0.370 g of a red solid. This material was purified via flash chromatography on a Biotage Isolera system (hexane:ethyl acetate gradient) with a silica column in which case a yellow solid (0.329 g, 71% yield) was obtained, which exhibited the following physical properties: mp 131–133°C; ¹H NMR (CDCl₃) δ 6.96 (d, J = 3.0 Hz, 1H), 4.46 (q, J = 6.0 Hz, 2H) and 1.42 (t, J = 6.0 Hz, 3H); ¹³C NMR (CDCl₃) δ 159.8, 127.1, 117.2, 111.3, 108.0, 107.0, 62.5 and 14.2; IR (neat) 2230 and 1692 cm⁻¹; HRMS (ES, M-H) m/z calcd for C₈H₇N₂O₂Br 240.9618, found 240.9632.

4.1.2 5-Cyano-4-(4-methoxyphenyl)-1H-pyrrole-2-carboxylic acid ethyl ester (8a)

Into a 20 mL microwave reaction vial equipped with a magnetic stirring bar was placed 4-bromo-5-cyano-1H-pyrrole-2-carboxylic acid ethyl ester (0.200 g, 0.823 mmol), 4-methoxyphenyl boronic acid (0.150 g, 0.987 mmol), DABCO (0.130 g, 1.15 mmol), dichloro[1,1′-bis-(diphenylphosphino)ferrocene]-palladium(II) dichloromethane adduct (0.031 g, 0.042 mmol) along with dioxane (6 mL) and water (6 mL). The reaction vial was capped and the mixture was heated in a Biotage Initiator microwave system for 2 hrs at 110°C. After cooling to room temperature, the crude reaction mixture was filtered through a short plug of silica gel and the plug was washed with an additional 25 mL of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give a dark brown residue. This material was purified via flash chromatography on a Biotage Isolera system (hexane:ethyl acetate gradient) with a silica column in which case a yellow solid (0.203 g, 91% yield) was obtained, which exhibited the following physical properties: mp 105–106°C; ¹H NMR (CDCl₃) δ 7.63 (d, J = 9.0 Hz, 2H), 7.09 (d, J = 3.0 Hz, 1H), 6.99 (d, J = 9.0 Hz, 2H), 4.43 (q, J = 6.0 Hz, 2H), 3.87 (s, 3H) and 1.43 (t, J = 6.0 Hz, 3H); ¹³C NMR (CDCl₃) δ 160.1, 159.8, 135.4, 128.0, 127.1,124.2, 114.5, 113.8, 112.9, 101.2, 61.8, 55.4 and 14.3; IR (neat) 2214 and 1679 cm⁻¹; HRMS (ES, M-H) m/z calcd for C₁₅H₁₃N₂O₃ 269.0932, found 269. 0979.

4.1.3 5-Cyano-4-(3,4-dimethoxyphenyl)-1H-pyrrole-2-carboxylic acid ethyl ester (8b)

This material was prepared in a manner identical to the previous example with the exception that 3,4-dimethoxyphenyl boronic acid was used as the boronic acid in which case a 100% yield of a light yellow solid was obtained, which exhibited the following physical properties: mp 145–147°C; ¹H NMR (CDCl₃) δ 7.23−7.25 (m, 2 Η), 7.06−7.08 (d, J = 3.0 Hz, 1H), 6.91 (d, J = 6.0 Hz, 1H), 4.45 (q, J = 6.0 Hz, 2H), 3.94 (s, 3H), 3.90 (s, 3H) and 1.41 (t, J = 6.0 Hz, 3H); ¹³C NMR (CDCl₃) δ 160.5, 149.3, 135.4, 127.0, 124.5, 119.4, 113.9, 112.9, 111.6, 109.9, 101.6, 62.0, 56.0 and 14.2: IR (neat) 2208 and 1720 cm⁻¹; HRMS (ES, M-H) m/z calcd for C₁₆H₁₅N₂O₄ 299.1037, found 299.1036.

4.1.4 5-Cyano-4-(3,4,5-trimethoxyphenyl)-1H-pyrrole-2-carboxylic acid ethyl ester (8c)

This material was prepared in a manner identical to the previous example with the exception that 3,4,5-trimethoxyphenyl boronic acid was used as the boronic acid in which case an 80% yield of a light yellow solid was obtained, which exhibited the following physical properties: mp 151–154°C; ¹H NMR (CDCl₃) δ 7.11 (s, 1H), 6.93 (s, 2H), 4.48 (q, J = 6.0 Hz, 2H), 3.94 (s, 6H), 3.91 (s, 3H), and 1.44 (t, J = 6.0 Hz, 3H); ¹³C NMR (CDCl₃) δ 160.3, 153.6, 138.4, 135.6, 127.1, 113.7, 113.0, 104.8, 104.1, 101.8, 62.0, 61.0, 56.3 and 14.3; IR (neat) 2217 and 1717 cm⁻¹; HRMS (ES, M-H) m/z calcd for C₁₇H₁₇N₂O₅ 329.1143, found 329.1153.

4.1.5 5-Cyano-4-(4-trifluoromethoxyphenyl)-1H-pyrrole-2-carboxylic acid ethyl ester (8d)

This material was prepared in a manner identical to the previous example with the exception that 4-trifluoromethoxyphenyl boronic acid was used as the boronic acid in which case an 62% yield of a white solid was obtained, which exhibited the following physical properties: mp 161–163°C; ¹H NMR (CDCl₃) δ 7.72 (d, J = 6.0 Hz, 2H), 7.32 (d, J = 6.0 Hz, 2H), 7.12 (s, 1H), 4.48 (q, J = 6.0 Hz, 2H) and 1.41 (t, J = 6.0 Hz, 3H); ¹³C NMR (CDCl₃) δ 160.6, 149.1, 133.8, 130.4, 128.2, 127.3, 121.5, 120.5 (q, J = 262.5 Hz), 113,3, 102.3, 62.3 and 14.2; IR (neat) 2218 and 1692 cm⁻¹; HRMS (ES, M-H) m/z calcd for C₁₅H₁₀N₂O₃F₃ 323.0649, found 323.0663.

4.1.6 5-Cyano-4-(3-methoxyphenyl)-1H-pyrrole-2-carboxylic acid ethyl ester (8e)

This material was prepared in a manner identical to the previous example with the exception that 3-methoxyphenyl boronic acid was used as the boronic acid in which case an 81% yield of a light brown solid was obtained, which exhibited the following physical properties: mp 137–139°C; ¹H NMR (CDCl₃) δ 7.39 (t, J = 6.0 Hz, 1H), 7.33–7.36 (m, 2H), 7.22 (s, 1H), 6.96–6.99 (m, 1H), 4.37 (q, J = 6.0 Hz, 2H), 3.88 (s, 3H) and 1.37 (t, J = 6.0 Hz, 3H); ¹³C NMR (CDCl₃) δ 160.7, 160.1, 135.1, 133.0, 130.1, 127.0, 119.2, 114.2, 113.6, 113.4, 112.1, 102.3, 62.2, 55.3 and 14.3: IR (neat) 2222 and 1688 cm⁻¹; HRMS (ES, M-H) m/z calcd for C₁₅H₁₃N₂O₃ 269.0932, found 269.0927.

4.1.7 5-Cyano-4-(4-methylphenyl)-1H-pyrrole-2-carboxylic acid ethyl ester (8f)

This material was prepared in a manner identical to the previous example with the exception that 4-methylphenyl boronic acid was used as the boronic acid in which case an 53% yield of a white solid was obtained, which exhibited the following physical properties: mp 155–158°C; ¹H NMR (CDCl₃) δ 7.59 (d, J = 6.0 Hz, 2H), 7.27 (d, J = 6.0 Hz, 2H), 7.11 (d, J = 3.0 Hz, 1H), 4.42 (q, J = 6.0 Hz, 2H), 2.41 (s, 3H) and 1.41 (t, J = 6.0 Hz, 3H); ¹³C NMR (CDCl₃) δ 160.5, 138.3, 135.5, 129.7, 128.8, 127.1, 126.6, 113.7, 113.2, 101.8, 62.0, 21.1 and 14.3; IR (neat) 2227 and 1687 cm⁻¹; HRMS (ES, M+H) m/z calcd for C₁₅H₁₅N₂O₂ 255.1128, found 255.1211.

4.1.8 4-(4-Chlorophenyl)-5-cyano-1H-pyrrole-2-carboxylic acid ethyl ester (8g)

This material was prepared in a manner identical to the previous example with the exception that 4-chlorophenyl boronic acid was used as the boronic acid in which case an 82% yield of a light yellow solid was obtained, which exhibited the following physical properties: mp 168–170°C; ¹H NMR (CDCl₃) δ 7.63 (d, J = 9.0 Hz, 2H), 7.43 (d, J = 9.0 Hz, 2H),7.11 (s, 1H), 4.46 (q, J = 6.0 Hz, 2H) and 1.44 (t, J = 6.0 Hz, 3H); ¹³C NMR (CDCl₃) δ 160.4, 134.3, 134.1, 130.1, 129.3, 128.0, 127.3, 113.3, 113.2, 102.0, 62.2 and 14.3; IR (neat) 2223 and 1689 cm⁻¹; HRMS (ES, M-H) m/z calcd for C₁₄H₁₀N₂O₂Cl 273.0436, found 273.0442.

4.1.9 5-Cyano-4-(3,4-dichlorophenyl)-1H-pyrrole-2-carboxylic acid ethyl ester (8h)

This material was prepared in a manner identical to the previous example with the exception that 3,4-dichlorophenyl boronic acid was used as the boronic acid in which case a 67% yield of a light yellow solid was obtained, which exhibited the following physical properties: mp 168–170°C; ¹H NMR (CDCl₃) δ 7.75 (m, 1H), 7.55–7.56 (m, 2H), 7.11 (s, 1H), 4.45 (q, J = 6.0 Hz, 2H) and 1.43 (t, J = 6.0 Hz, 3H); ¹³C NMR (CDCl₃) δ 159.5, 133.3, 133.0, 132.6, 131.6, 131.0, 128.6, 127.6, 125.9, 113.1, 112.8, 101.8, 61.9 and 14.3; IR (neat) 2227 and 1688 cm⁻¹; HRMS (ES, M-H) m/z calcd for C₁₄H₉N₂O₂Cl₂ 307.0047, found 307.0053.

4.1.10 5-Cyano-4-phenyl-1H-pyrrole-2-carboxylic acid ethyl ester (8i)

This material was prepared in a manner identical to the previous example with the exception that phenyl boronic acid was used as the boronic acid in which case a 66% yield of a light yellow solid was obtained, which exhibited the following physical properties: mp 179–182°C; ¹H NMR (CDCl₃) δ 7.71 (d, J = 6.0 Hz, 2H), 7.47 (t, J = 6.0 Hz, 2H), 7.41 (t, J = 6.0 Hz, 1H), 7.14 (d, J = 3.0 Hz, 1H), 4.45 (q, J = 6.0 Hz, 2H) and 1.43 (t, J = 6.0 Hz, 3H); ¹³C NMR (CDCl₃) δ 160.1, 135.5, 131.6, 129.0, 128.3, 127.2, 126.8, 113.5, 113.3, 101.8, 61.9 and 14.3: IR (neat) 2221 and 1682 cm⁻¹; HRMS (ES, M+H) m/z calcd for C₁₄H₁₃N₂O₂ 241.0972, found 241.1030.

4.1.11 5-Cyano-4-(4-methoxycarbonylphenyl)-1H-pyrrole-2-carboxylic acid ethyl ester (8j)

This material was prepared in a manner identical to the previous example with the exception that 4-carbomethoxyphenyl boronic acid was used as the boronic acid in which case a 63% yield of a light yellow solid was obtained, which exhibited the following physical properties: mp 208–210°C; ¹H NMR (CDCl₃) δ 8.13 (d, J = 9.0 Hz, 2H), 7.78 (d, J = 9.0 Hz, 2H), 7.19 (s, 1H), 4.44 (q, J = 6.0 Hz, 2H), 2.96 (s, 3H) and 1.43 (t, J = 6.0 Hz, 3H); ¹³C NMR (CDCl₃) δ 166.7, 160.0, 136.0, 134.1, 130.4, 129.8, 127.5, 126.6, 113.4, 113.2, 102.3, 62.1, 52.3 and 14.3; IR (neat) 2223 and 1721 and 1694 cm⁻¹; HRMS (ES, M-H) m/z calcd for C₁₆H₁₄N₂O₄ 298.0959, found 298.0921.

4.1.12 4-(4-Acetylphenyl)-5-cyano-1H-pyrrole-2-carboxylic acid ethyl ester (8k)

This material was prepared in a manner identical to the previous example with the exception that 4-acetylphenyl boronic acid was used as the boronic acid in which case an 87% yield of a light brown solid was obtained, which exhibited the following physical properties: mp 198–200°C; ¹H NMR (CDCl₃) δ 8.06 (d, J = 6.0 Hz, 2H), 7.80 (d, J = 6.0 Hz, 2H), 7.20 (d, J = 3.0 Hz, 1H), 4.43 (q, J = 6.0 Hz, 2H), 2.65 (s, 3H) and 1.43 (t, J = 6.0 Hz, 3H); ¹³C NMR (CDCl₃) δ 197.5, 159.9, 136.6, 136.1, 134.0, 129.1, 127.6, 126.8, 113.4, 113.2, 102.3, 62.0, 26.6 and 14.3; IR (neat) 2366 and 1712 and 1680 cm⁻¹; HRMS (ES, M-H) m/z calcd for C₁₆H₁₃N₂O₃ 281.0932, found 281.0933.

4.1.13 5-Cyano-4-furan-3-yl-1H-pyrrole-2-carboxylic acid ethyl ester (8l)

This material was prepared in a manner identical to the previous example with the exception that 3-furanylboronic acid was used as the boronic acid in which case a 75% yield of a light yellow solid was obtained, which exhibited the following physical properties: mp 117–119°C; ¹H NMR (CDCl₃) δ 7.89 (dd, J = 1.5 Hz, J = 3.0 Hz, 1H), 7.51 (t, J = 3.0 Hz, 1H), 6.99 (d, J = 1.5 Hz, 1H), 6.79 (dd, J = 1.5 Hz, J = 3.0 Hz, 1H), 4.41 (q, J = 6.0 Hz, 2H), and 1.42 (t, J = 6.0 Hz, 3H); ¹³C NMR (CDCl₃) δ 160.4, 143.7, 139.6, 127.3, 127.2, 117.4, 113.4, 112.6, 108.7, 101.6, 62.0 and 14.3; IR (neat) 2223 and 1738 cm⁻¹; HRMS (ES, M+H) m/z calcd for C₁₂H₁₁N₂O₃ 231.0764, found 231.0746.

4.1.14 5-Cyano-4-thiophen-3-yl-1H-pyrrole-2-carboxylic acid ethyl ester (8m)

This material was prepared in a manner identical to the previous example with the exception that 3-thiophenylboronic acid was used as the boronic acid in which case a 96% yield of a light brown solid was obtained, which exhibited the following physical properties: mp 142–145°C; ¹H NMR (CDCl₃) δ 7.67 (dd, J = 1.5 Hz, J = 3.0 Hz, 1H), 7.47 (dd, J = 1.5 Hz, J = 6.0 Hz, 1H), 7.43 4. (dd, J = 3.0 Hz, J = 6.0 Hz, 1H), 7.08 (d, J = 3.0 Hz, 1H), 4.44 (q, J = 6.0 Hz, 2H), and 1.43 (t, J = 6.0 Hz, 3H); ¹³C NMR (CDCl₃) δ 160.5, 132.5, 130.5, 127.1, 126.5, 126.0, 121.8, 113.7, 113.0, 101.7, 62.1 and 14.3; IR (neat) 2230 and 1679 cm⁻¹; HRMS (ES, M-H) m/z calcd for C₁₂H₉N₂O₂S 245.0324, found 245.0393.

4.1.15 5-Cyano-4-(2,3,4-trimethoxyphenyl)-1H-pyrrole-2-carboxylic acid ethyl ester (8n)

This material was prepared in a manner identical to the previous example with the exception that 2,3,4-trimethoxyphenylboronic acid was used as the boronic acid in which case a 70% yield of a yellow solid was obtained, which exhibited the following physical properties: mp 106–107°C; ¹H NMR (CDCl₃) δ 7.23 (d, J = 6.0 Hz, 1H), 7.14 (d, J = 3.0 Hz, 1H), 6.75 (d, J = 6.0 Hz, 1H), 4.46 (q, J = 6.0 Hz, 2H), 3.93 (s, 3H), 3.92 (s, 3H), 3.84 (s, 3H) and 1.42 (t, J = 6.0 Hz, 3H); ¹³C NMR (CDCl₃) δ 160.9, 154.0, 151.5, 142.6, 131.6 126.6, 124.2, 118.7, 115.7, 113.6, 107.6, 103.7, 61.9, 60.0, 56.0 and 14.3; IR (neat) 2221 and 1679 cm⁻¹; HRMS (ES, M+Na) m/z calcd for C₁₇H₁₈N₂O₅Na .253.1108, found 253.1100.

4.1.16 3-Bromo-5-cyano-4-(2,3,4-trimethoxyphenyl)-1H-pyrrole-2-carboxylic acid ethyl ester (9)

Into a 100 mL round bottom flask equipped with magnetic stirring bar was placed 5-cyano-4-(2,3,4-trimethoxyphenyl)-1H-pyrrole-2-carboxylic acid ethyl (0.250g, 0.756 mmol), potassium hydroxide (0.040g, 0.0756 mmol) and DMF (10 mL) and the reaction mixture was capped and stirred for 30 mins. NBS (0.270g, 1.51 mmol) dissolved in in 5 mL of DMF was added slowly to the reaction mixture and the resulting solution was capped and stirred for 24 hrs. The reaction mixture was subsequently diluted with 20 mL of a 20% solution of sodium thiosulfate and 15 mL of ethyl acetate was also added. The phases were separated and the aqueous phase was extracted with addition ethyl acetate (2 × 15 mL) and the combined organic phases were washed with 20 mL of a saturated aqueous lithium chloride solution followed by drying over anhydrous sodium sulfate. After removing the drying agent by filtration and concentrating the ethyl acetate solution in vacuo, an orange solid (86% yield) was obtained, which was very pure. An analytical sample was prepared by flash chromatography on a Biotage Isolera system (hexane:ethyl acetate gradient) with a silica column in which case a yellow solid was obtained and exhibited the following physical properties; mp 173–176 °C; ¹H NMR (CDCl₃) δ 6.97 (d, J = 8.4 Hz, 1H), 6.75 (d, J = 8.4 Hz, 1H), 4.45 (q, J = 7.2 Hz, 2H), 3.93 (s, 6H), 3.83 (s, 3H) and 1.45 (t, J = 7.2 Hz, 3H); ¹³C NMR (CDCl₃) δ 159.2, 154.8, 152.0, 142.2, 133.0, 126.0, 124.2, 116.3, 111.9, 106.9, 104.9, 104.8. 62.1, 61.2, 61.1, 56.0 and 14.2; IR (neat) 2221 and 1729 cm⁻¹; HRMS (ES, M+H) m/z calcd for C₁₇H₁₈N₂O₃Br 409.0230, found 409. 0283.

4.1.17 4-(2,3,4-trimethoxyphenyl)-5-formyl-1H-pyrrole-2-carboxylic acid Ethyl Ester (10)

To a 20 mL microwave vial was added 4-bromo-5-formyl pyrrole 2-carboxylic acid ethyl ester (0.5 g, 2.032 mmol), 2,3,4-trimethoxyphenyl trifluoroborate (0.723g, 2.64 mmol), Pd-tetrakis-(triphenylphosphine) (0.023g, 0.02 mmol), Hunig’s base (0.341g, 2.64 mmol) in 3:1 toluene; ethanol with 20 drops of water. The reaction mixture was microwaved for 110°C for 2 hours. After cooling the reaction mixture to room temperature, it was filtered through a short silica plug and the resulting mixture was evaporated in vacuo. The crude product was dried using a Kulgelrohr apparatus to give a reddish brown solid (0.75g). The crude residue was subjected to flash chromatography on a Biotage SP-1 instrument with a silica column in which case 0.51 g (75% yield) of a dark brown solid was obtained upon elution with seven column volumes of hexane/ethyl acetate gradient. This solid exhibited the following properties: m.p. 138–140 °C; 1H NMR (acetone-d₆, 500 MHz) δ 1.36 (t, J = 7.2 Hz, 3H), 3.66 (s, 3H), 3.88 (s, 3H), 3.91 (s, 3H), 4.36 (q, J = 7.2 Hz, 2H), 6.89 (d, J = 8.5 Hz, 1H), 6.96 (s, 1H), 7.11 (d, J = 8.5 Hz, 1H), 9.63 (s, 1H); 13C NMR (acetone-d6, 75 MHz) δ 180.3, 159.0, 154.6, 152.0, 142.4, 130.9, 130.7, 126.7, 124.6, 117.1, 107.5, 104.9, 60.9, 60.4, 60.2, 55.5, 13.6; IR (neat) 1709 and 1660 cm-1; HRMS (ES) m/z calcd for C₁₇H₁₉NO₆ [M+Na] 356.1105, found 356.1077.

4.1.18 3-Bromo-4-(2,3,4-trimethoxyphenyl)-5-formyl-1H-pyrrole-2-carboxylic acid Ethyl Ester (11)

To a 100 mL round bottom flask equipped with a stir bar, was added 4-(2,3,4-trimethoxyphenyl)-5-formyl pyrrole 2-carboxyic acid ethyl ester (0.100 g, 0.300 mmol) and potassium hydroxide (0.034 g, 0.6 mmol) in 15 mL of DMF. The reaction mixture was allowed to stir for 15 minutes at room temperature, after which N-bromo succinimide (0.053 g, 0.33 mmol) was added and the reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with 30 mL water and 15 mL of sodium thiosulfate solution was added to the reaction mixture. The reaction mixture was extracted with ethyl acetate (3 × 15 mL). The combined ethyl acetate layers were dried over anhydrous sodium sulfate, filtered and concentrated to give a dark brown solid (0.12 g, 96% yield). This solid exhibited the following properties: m.p. 158–160 °C; ¹H NMR (CDCl₃) δ 1.43 (t, J = 7.2 Hz, 3H), 3.68 (s, 3H), 3.93 (s, 3H), 3.94 (s, 3H), 4.45 (q, J = 7.2 Hz, 2H), 6.81 (d, J = 8.5 Hz, 1H), 7.04 (d, J = 8.5 Hz, 1H), 9.45 (s, 1H); ¹³C NMR (CDCl₃) δ 181.0, 159.5, 154.5, 151.8, 142.3, 131.2, 130.2, 126.7, 124.6, 116.7, 107.2, 105.3, 61.6, 60.2, 56.0, 14.2; IR (neat) 1708 and 1660 cm⁻¹; HRMS (ES, M+Na) m/z calcd for C₁₇H₁₈NNaBrO₆ 434.0210, 436.0192 found 434.0199, 436.0186.

4.1.19 (E/Z)-3-Bromo-4-(2,3,4-trimethoxyphenyl)-5-oximino-1H-pyrrole-2-carboxylic acid Ethyl Ester (12)

To a 100 mL round bottom flask equipped with a stir bar, was added 4-(2′,3′,4′-trimethoxyphenyl)-5-formyl-pyrrole-2-ethyl ester (0.13 g, 0.315 mmol), hydroxyl amine hydrochloride (0.022 g, 0.315 mmol) and 0.1 mL of pyridine into 15 mL of ethanol. The reaction mixture was allowed to reflux for 2 hours. The reaction mixture was cooled and concentrated in vacuo to give a crude residue. Water (10 mL) was added to the residue and the solution was cooled in an ice bath and stirred until the oxime crystallized. The solid was filtered and washed with water (2 × 15 mL) and dried to give (E/Z)-3-bromo-4-(2,3,4-trimethoxyphenyl)-5-oximino-1H-pyrrole-2-carboxylic acid ethyl ester (NT-7-45, 12), as an orange solid (0.091g, 68 %).This solid mixture was subjected to flash chromatography on Biotage Isolera instrument with a silica column with an ethyl acetate/hexane gradient in which case 0.040 g (30% yield) of the Z isomer (12a) and 0.045 g (34% yield) of the E isomer (12b) were obtained.

(Z)-3-Bromo-4-(2,3,4-trimethoxyphenyl)-5-oximino-1H-pyrrole 2-carboxylic acid ethyl ester (12a) exhibited the following properties: m.p. 141–143 °C; ¹H NMR (acetone-d₆) δ 1.39 (t, J = 7.2 Hz, 3H), 3.64 (s, 3H), 3.86 (s, 3H), 3.91 (s, 3H), 4.39 (q, J = 7.2 Hz, 2H), 6.87 (d, J = 8.4 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H) and 7.03 (s, 1H); ¹³C NMR (acetone-d₆) δ 159.1, 154.2, 152.2, 142.4, 139.4, 127.0, 126.7, 124.7, 120.9, 118.4, 107.4, 105.2, 60.2, 60.1, 55.4 and 13.7; IR (neat) 1710 cm⁻¹; HRMS (ES, M+Na) m/z calcd for C₁₇H₁₉N₂NaBrO₆ 449.0319, 451.0301; found 449.0314, 451.0296.

(E)-3-Bromo-4-(2,3,4-trimethoxyphenyl)-5-oximino-1H-pyrrole 2-carboxylic acid ethyl ester (12b) exhibited the following properties: m.p. 125–126 °C; ¹H NMR (acetone-d₆) δ 1.39 (t, J = 7.2 Hz, 3H), 3.64 (s, 3H), 3.86 (s, 3H), 3.91 (s, 3H), 4.39 (q, J = 7.2 Hz, 2H), 6.88 (d, J = 8.4 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 7.74 (s, 1H); ¹³C NMR (acetone-d₆) δ 159.1, 154.2, 152.2, 142.4, 139.4, 127.1, 126.7, 124.8, 121.0, 118.4, 107.5, 105.2, 60.3, 60.1, 55.4 and 13.7; IR (neat) 1710 cm⁻¹; HRMS (ES, M+Na) m/z calcd for C₁₇H₁₉N₂NaBrO₆ 449.0319, 451.0301; found 449.0313, 451.0297.

4.2 Biological Evaluations

The HeLa, SK-OV-3 and A-10 cell lines were obtained from the ATCC (Manassas, VA) and MDA-MB-435 cells were from the Lombardi Cancer Center of Georgetown University. The antiproliferative effects of the compounds were evaluated using the SRB assay¹⁰ as previously described.¹¹ Descriptions of the generation, characterization and culture conditions of the HeLa WTβIII and SK-OV-3 MDR1-M6/6 cell lines were reported previously.¹¹ The microtubule depolymerizing effects were evaluated in A-10 cells as described previously.⁷ For the in vivo testing, six-week old athymic nude female mice were implanted bilaterally with MDA-MB-435 cells. When tumors reached an average size of 200 mm,³ paclitaxel (20 mg/kg/dose) on days 0, 2, 4, 6, 8 and 11 or NT-7-45 (12) (75 mg/kg/dose) daily on days 0–8 was initiated. Paclitaxel was prepared in 50:50 mixture of ethanol: cremophor and then diluted into PBS and NT-7-45 was solubilized in a mixture of 2:1 Tween80: DMSO and then diluted into PBS. Mice were observed and weighed twice a week to monitor potential weight loss as a sign of toxicity. Tumors from the control and NT-7-45 treated groups were removed at the end of the trial, day 14, and weighed and individual masses recorded. The average tumor volume data in panel A were analyzed using 2-way ANOVA followed by Dunnett’s post-hoc test to correct for multiple comparisons. The final tumor weights shown in panel B were evaluated using an unpaired, two-tailed Student’s t-test. The mouse studies were conducted in accordance with NIH guidelines in an AALAC approved facility with food and water provided ad libitum.

4.3 Stastical Analyses

The average tumor volumes of 10 tumors in each treatment group in the in vivo trial, shown in Figure 3A, and including other agents not shown here, were compared to the average tumor volume of the control group over time using a 2-way ANOVA followed by Dunnett’s post-hoc test to correct for multiple comparisons. The final tumor weights shown in Figure 3, panel B were evaluated using an unpaired, two-tailed Student’s t-test.

4.4 Molecular Modeling

The X-ray crystal structure of αβ-tubulin complexed with DAMA-colchicine (pdbid: 1SA0) was prepared with Sybyl 8.1.¹⁵ The stathmin-like domain and the C and D subunits were deleted. Hydrogen atoms were added and their orientations were optimized by the Tripos force field to a gradient of 0.005 kcal mol⁻¹ Å⁻¹. The docking studies were performed using GOLD 5.0.¹² The ligands were docked in the active site, which was defined by the space in a 6 Å radius around DAMA-colchicine. Docking conformations for KL-3-95 generated with GOLD and filtered initially by GoldScore were further analyzed with HINT.¹⁴ The resulting conformations were rescored with HINT and the best docking pose was used as the binding pose.