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. Author manuscript; available in PMC: 2011 Nov 25.
Published in final edited form as: J Med Chem. 2010 Oct 25;53(22):8129–8139. doi: 10.1021/jm101024j

An Assessment of Chemoselective Neoglycosylation Methods Using Chlorambucil as a Model

Randal D Goff 1, Jon S Thorson 1,*
PMCID: PMC2988911  NIHMSID: NIHMS248350  PMID: 20973561

Abstract

To systematically assess the impact of glycosylation and the corresponding chemoselective linker upon the anticancer activity/selectivity of the drug chlorambucil, herein we report the synthesis and anticancer activities of a 63-member library of chlorambucil-based neoglycosides. A comparison of N-alkoxyamine-, N-acyl hydrazine- and N-hydroxyamine-based chemoselective glycosylation of chlorambucil revealed sugar-and linker-dependent partitioning among open and closed-ring neoglycosides and corresponding sugar-dependent variant biological activity. Cumulatively, this study represents the first neoglycorandomization of a synthetic drug and expands our understanding of the impact of sugar structure upon product distribution/equilibria in the context of N-alkoxyamino-, N-hydroxyamino- and N-acyl hydrazine-based chemoselective glycosylation. This study also revealed several analogs with increased in vitro anticancer activity, most notably D-threoside 60 (NSC 748747), which displayed much broader tumor specificity and notably increased potency over the parent drug.

Introduction

The sugars attached to pharmaceutically important natural products dictate the pharmacokinetics and/or pharmacodynamics of the selected agent.1–3 Yet, studies designed to systematically understand and/or exploit the attachment of carbohydrates in drug discovery remain limited by the availability of practical synthetic and/or biosynthetic tools.4–8 Neoglycosylation takes advantage of a chemoselective reaction between free reducing sugars and N-methoxyamino-substituted acceptors.9–19 This reaction has enabled the process of `neoglycorandomization' wherein alkoxyamine-appended natural product-based drugs are differentially glycosylated with a wide array of natural and unnatural reducing sugars.4–8,20–24 Neoglycorandomization has led to the discovery of cardenolide neoglycosides with enhanced in vitro and in vivo anticancer activity and lower in vivo toxicity,20,23 colchicine neoglycosides with a novel anticancer mechanism and lower in vivo toxicity,21 vancomycin neoglycosides which displayed improved in vitro potency against clinical isolates of vancomycin-resistant Enterococci,22 and betulinic acid neoglycosides improved for either in vitro anticancer or antiviral potency.24 While these examples clearly highlight the potential impact of differential glycosylation in drug lead discovery, this work has not addressed the impact of the chemoselective glycosylation `handles'17 employed.

To assess the impact of alternative chemoselective glycosylation methods in the context of neoglycorandomization, we selected the synthetic anticancer agent chlorambucil (Figure 1, 1) as a model. First synthesized over five decades ago,25 chlorambucil remains a current treatment for chronic lymphocytic leukemia (CLLa)26–30 and has served as the basis for newer generation analogs such as the recently approved bendamustine (3).31 A nitrogen mustard, 1 leads to guanine alkylation and DNA cross-linking, and ultimately prohibits DNA replication and transcription.32–33 The primary cellular uptake mechanism of 1 is passive diffusion34 and like many cytotoxics, the lack of nitrogen mustard tumor-specificity contributes to serious side effects.35,36 Thus, improvements have focused upon i) the development of tumor-activated prodrugs – exemplified by the hypoxia-activated N-oxide PX-478 (Figure 1, 2) currently in phase I;37 or ii) modifications to engage tumor-specific transport – exemplified by the β-D-glucosyl analog of ifosfamide (glufosfamide, Figure 1, 4) which is actively transported into tumor cells by the sodium/D-glucose cotransporter SGLT3 (SAAT1).38 While the specific glycosylation of 1 has presented analogs which display slight improvements in a perceived therapeutic index (slightly improved in vitro potency and subtle reductions of in vivo peripheral toxicity), the analogs synthesized to date have been restricted to the use of D-gluco or D-galacto-based sugars.39–46

Figure 1.

Figure 1

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Chlorambucil (1) and other nitrogen mustard compounds including N-oxide 2 (PX-478), FDA-approved bendamustine (3), and glycosylated variants glufosfamide (4) and 5. The latter was found to inhibit the brain/erythrocyte D-glucose GLUT1 transporter and thereby decrease glucose uptake (reference 42).

To systematically assess the impact of glycosylation and the corresponding chemoselective linker upon the anticancer activity/selectivity of 1, herein we report the synthesis and anticancer activities of a 54-member library of 1-neoglycosides. Several analogs with increased in vitro anticancer activity were identified, most notably D-threoside 60 (NSC 748747) which displayed much broader tumor specificity and increases in potency of up to 15-fold compared to 1 – representing the most active chlorambucil glycoside reported to date. Representative sugars identified as hits in the context of N-methoxyamino-substituted 1, were subsequently conjugated via N-acyl hydrazine- and N-hydroxyamine-based strategies and the products characterized and evaluated for anticancer activity. Analysis of these second generation neoglycoside analogs revealed sugar-dependent partitioning among open and closed-ring neoglycosides and corresponding sugar-dependent variant biological activity. Cumulatively, this study represents the first neoglycorandomization of a synthetic drug and expands our understanding of the impact of sugar structure upon product distribution/equilibria in the context of N-alkoxyamino-, N-hydroxyamino- and N-acyl hydrazine-based chemoselective glycosylation.

Results and Discussion

Chlorambucil N-alkoxyamino-based neoglycorandomization (Scheme 1)

Scheme 1.

Scheme 1

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Synthesis of the N-alkoxyamino-based chlorambucil librarya

a(a) MeON(H)Me, NMM, EDAC, 0 °C (97%); (b) LAH, THF, 0 °C (70%); (c) MeONH3Cl, Et3N, EtOH (84%); (d) BH3·Et3N, HCl, EtOH, 0 °C (65%); (e) reducing sugar, MeOH, HOAc (1.5 eq.), 40 °C (63% avg.).

Chlorambucil was converted to the Weinreb amide 6 using the EDAC coupling agent in excellent yield (97%) and then selectively reduced to the corresponding aldehyde with LAH. Compound 7 was subsequently condensed with methoxyamine•HCl in the presence of organic base providing a mixture of E- and Z-oximes (8). Reduction of the carbon-nitrogen double bond was accomplished with BH3•Et3N in a manner similar to that for previously described neoaglycons.20,21,23 This convenient strategy yielded neoaglycon 9 in four steps with an overall yield of 37% and required only a single chromatographic purification. While standard neoglycosylation conditions (3/1 DMF/HOAc)9–22 provided a slightly greater yield (68%) of neo-D-riboside 53 in a pilot reaction, the use of MeOH with a small molar excess of HOAc (i.e., 1.5 equivalents) as an acidic proton source was sufficient for the reaction to proceed in an equivalent amount of time (54% yield, Table S1, Supporting Information) and also simplified subsequent solvent evacuation. Using these optimized conditions (90 μM 9, 2 equiv. sugar, 40 °C, 1.5 eq. HOAc in MeOH) a 54-member library of neoglycosides (10–63, see Figure S1, Supporting Information) was synthesized with an average isolated yield of 63% wherein high-throughput solid phase extraction provided an average purity of 92.9% (see Table S3, Supporting Information). Product yields paralleled the overall reactivity trend: tetroses (79±10%) > deoxy sugars (69±6%) > pentoses (62±7%) > hexoses (56±5%) – likely reflecting the general reducing sugar solubility in the selected solvent system. Unlike prior neoglycorandomized libraries,9–24 a preference for β-anomer formation of chlorambucil neoglycosides was generally less predominate. Rather, a strong 1,2-trans relationship was typically observed (Table S2, Supporting Information).

Anticancer activity of chlorambucil N-alkoxyamino-based neoglycosides

The antiproliferative activity (i.e., GI50) of neoglycosides 10–63 were evaluated using a ten-member panel of human carcinomas from various lung, colorectal, liver, breast, prostate, CNS, and ovarian cell lines with 1 and neoaglycon 9 as comparators (see Figure 2). Based upon this study, aglycon 9 displayed slightly better GI50 values (1.1–4.4 μM) compared to the parent drug 1 (5.0–11.0 μM) and the vast majority of the library members had an average GI50 also comparable to or lower than chlorambucil (see Table S5, Supporting Information). Out of the 54 analogs, 19 possessed GI50s in the high nanomolar range in at least one cell line, six of which displayed GI50s in the high nanomolar range in three or more lines. Of this latter set, two neoglycosides (D-glucuronolactonide 39 and D-threoside 60, see Figure 3) were identified as the two most potent neoglycosides. In comparison to the parent 1, 39 displayed a 6-fold improvement in average growth inhibition across the cell panel, with the most sensitive cell line being SF268 glioblastoma (12-fold improved). Likewise, D-threoside 60 presented an average elevated potency of 8-fold over 1, with 12-, 13-, and 15-fold improved activities toward HT29 (colorectal), H1299 (lung), and HCT15 (colorectal) cancers, respectively, over the parent drug.

Figure 2.

Figure 2

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Summary of GI50 data from the high-throughput growth inhibition assay of 10–63 (reciprocal values displayed). Comparisons were performed against the aglycon (9) and chlorambucil (1). GI50 data and error values are provided in the Supporting Information, Table 5. Representative cancer cell lines tested include: NCI-H460 (lung), A549 (lung), Du145 (prostate), SKOV3 (ovary), Hep3b (liver), SF268 (brain), MCF7 (breast), HT29 (colorectal), HCT15 (colorectal), H1299 (lung).

Figure 3.

Figure 3

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Structures of the most antiproliferative chlorambucil N-alkoxyamino-based neoglycosides against a ten-member carcinoma panel.

The activity assessment described above revealed the following general structure-activity relationships. First, sugars that favor furanosyl-derived neoglycosides (e.g., threosides 60 & 61, glucuronolactonide 39, arabinosides 14 & 15, lyxosides 41 & 42, and xylosides 62 & 63) led to the greatest improvements in anticancer activity. Second, relative configuration within this furanoside-derived neoglycoside series influenced potency. Specifically, those tetroses or pentoses with a 2,3-trans dihydroxy orientation [e.g., D-threoside 60 (2S,3R), L-threoside 61 (2R,3S), and 39 (D-2S,3R)] were generally more potent than those with a corresponding 2,3-cis configuration [e.g., D-erythroside 19 (2R,3R), D-riboside 53 (2R,3R), and L-riboside 54 (2S,3S)] while D- and L-saccharide enantiomers of this furanoside-derived group were found to have similar values. Third, among neohexosides, a 3R hydroxyl group (i.e., axial in the chair conformation) enhances selectivity toward lung (H1299) and colon (HCT-15) cancer cell lines (e.g., allosides 10 & 11, altrosides 12 & 13, and L-guloside 40). Finally, most neoglycosides deriving from sugars known to be GLUT substrates and mediate GLUT-dependent uptake of conjugates (e.g., D-glucoside 28 or 3-methoxy-D-glucoside 33)47–50 were not among the most active and/or selective hits identified. Additionally, comparison of the anomeric composition of either the most active neoglycosides or the library as a whole to the inhibitory data does not reveal that a distinct correlation exists between anomers and activity. To illustrate using the antiproliferative neo-D-pentosides (the two with the most extreme anomeric biases), D-arabinoside 14 (α/β 19/1) and D-xyloside 62 (β only) have similar GI50s in nine of the ten cell lines, not providing any distinction between the two anomers and overall impact on growth inhibition.

Chlorambucil N-hydroxyamino-based neoglycosylation (Scheme 2)

Scheme 2.

Scheme 2

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Synthesis of the N-acyl hydrazine- and N-hydroxyamino-based chlorambucil librariesa

a(a) HONH3Cl, Et3N, EtOH (98%); (b) BH3·Et3N, HCl, EtOH, 0 °C (65%); (c) reducing sugar, MeOH, HOAc (1.5 eq.), 40 °C (47% avg.); (d) (i) N-hydroxysuccinimide, DIC, THF, 40 °C, (ii) NH2NH2, pyridine, DMAP, 40 °C (85%); (e) reducing sugar, MeOH, HOAc (1.5 eq.), 40 °C (62% avg.).

Given the clear impact of chlorambucil neoglycosylation upon anticancer activity, we subsequently set out to examine the specific contribution of the neoglycoside handle. Similar to alkoxyamino-based chemoselective neoglycosylation,9–24 previous studies have revealed hydroxyamines51–53 and hydrazides54–59 to also provide the corresponding closed-ring glycosides. Additionally, we envisioned that use of a hydroxyamino handle may allow for additional modification at the hydroxyl group, providing a facile means for further diversification.23 To create the two modified aglycons, only slight changes in the synthesis were required. In a fashion similar to the procedure described in Scheme 1, aldehyde 7 was combined with hydroxyamine HCl to form a mixture of E- and Z-oximes (64) that were reduced with BH3•Et3N complex. As with the synthesis described in Scheme 1, only aglycon 65 required column chromatography for purification in the four-step process from 1 (Scheme 2, 44% overall yield). Hydrazide formation (71) was achieved by reacting the N-hydroxysuccinimidyl ester of 1 with hydrazine in the presence of DMAP in pyridine. Using the previously described neoglycosylation conditions (see Scheme 1), the focused hydroxyamine (66–70) and hydrazide (72–75) neoglycosyl sets were synthesized using sugars identified as hits from the alkoxyamino-based series 10–63 – specifically: D-glucuronolactone and D-threose, which had superior performance throughout the panel; D-xylose, the most active of the pentoses; and D-fucose, which displayed the most pronounced selectivity toward one tumor line (HCT-15 colorectal).

NMR analysis of the 66–70 neoglycosides revealed the desired closed-ring α- and β-anomeric forms were usually in equilibrium with an open-chain imine isomer. Evidence for this was based upon the observed 1H NMR chemical shift of the H1 (doublets) and H2 (doublets of doublets) protons at 7.2–7.5 and 5.1–5.0 ppm, respectively, indicating the presence of an iminyl double-bond. Such nitrone formation is well-precedented for the condensation of aldehydes and monosubstituted hydroxyamines.60 However, in the context of glycoside formation, only a handful of such glycosides have been reported, comprised of protected saccharides and small N-alkyl-N-hydroxyamines.51–53 From the current study, the nature of the sugar influences the thermodynamic equilibrium and thereby, product distribution. Specifically, the D-fucoside-derived 66 predominately adopted the closed-ring isomer (67%), D-glucuronolactone (67) and D-ribose (68) favored the open-chain nitrone (71% and 50%, respectively), while D-threose (70) led solely to nitrone (Figure 4; Table S3, Supporting Information). These equilibria could be shifted upon nitrone modification. For example, peracetylation of neoriboside 68 using acetic anhydride and DMAP in THF promoted ring closure based upon 1H NMR (Figure 5) and ESI-MS analysis. Deacetylation of the peracetate 69 in base reestablished the mixture of isomers, demonstrating that changing the electronics of the nitrone oxygen will promote ring opening and closure, as will the type of ligated sugar. Interestingly, the anomeric ratio also changed, moving from a 1/1 ratio as the nitrone to 2/1 as the tetraacetate, contrasting from the 1/3 α/β ratio of methoxyamine D-riboside (53).

Figure 4.

Figure 4

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1H NMR spectra of N-hydroxyaminochlorambucil glycosides. A. Equilibrium between the cyclic neoglycoside and acyclic nitrone of D-fucoside 66. B. Nitrone form of the chlorambucil D-threoside 70. Both spectra obtained at 500 MHz in CD3OD.

Figure 5.

Figure 5

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Interconversion of N-hydroxyaminochlorambucil-D-riboside between the cyclic neoglycoside and acyclic nitrone 68 and peracetylated analog 69. Both spectra obtained at 500 MHz with 68 in CD3OD and 69 in CDCl3.

Chlorambucil N-acyl hydrazine-based neoglycosylation (Scheme 2)

A conformational study of sugar acetylhydrazides by Bendiak indicated that natural hexose and pentose (e.g., D-Glc, D-Gal, D-Xyl, etc.) analogs formed pyranosides, as evidenced by 1H NMR.54 Similar findings were reported for mono- and oligosaccharide chemoselective ligations with biotinyl,55 long-chain acyl,56 peptidyl,57 and adipyl hydrazides.59 In contrast, the current chlorambucil study revealed a trend that mirrored that which was observed for the 66–70 subset. Specifically, D-fucoside 72 was formed only as the cyclic compound (67% yield), D-glucuronolactonide 73 and D-xyloside 75 were a mixture of the open and closed conformers (46% and 47%, respectively), and D-threoside 74 adopted the open-chain imine (86%). These results indicate that closure of the glycoside may not only be dependent on the type of sugar but possibly the nature of the aglycon as well. It was also notable that only the β-anomer of the closed-rings was observed while the hydrazyl imines were isolated as mixtures of E- and Z-isomers (Figure 6).

Figure 6.

Figure 6

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1H NMR spectra of N-hydrazidochlorambucil glycosides. A. Equilibrium between the cyclic neoglycoside and acyclic imine of D-glucurono-6,3-lactonide 73. B. Nitrone form of the chlorambucil D-threoside 74. Both spectra obtained at 500 MHz in CD3OD.

Anticancer activity of chlorambucil N-hydroxyamino- and N-acyl hydrazine-based neoglycosides

Neoglycosides 66–70, 72–75, and their respective aglycons, 65 and 71, were assayed for antiproliferative activity using the same cell lines as 10–63. In general, the alternate handle glycosides did not perform as well as their methoxyamine analogs with the most notable difference being between the 66–70 and 72–75 analogs. While some of the hydroxyamine compounds produced comparable inhibitory responses to the methoxyamine group, 72–75 had universally diminished anticancer properties over 10–63 with average GI50 values 5- to 13-fold greater across the ten-member panel. 66–70 and 72–75 were also found to have GI50 values up to 6-fold larger than the corresponding aglycons, though aglycons 65 and 71 were of similar activity to 9 (Table S5, Supporting Information).

Two exceptions to this overall trend, D-riboside 68 and its peracetylated variant 69, were 2- to 5-fold more active than the alkoxyamino-based 53 in six of the ten cell lines (0.53–1.3 μM vs. 1.8–5.9 μM, respectively). 68 and 69 are also notable as among the most active (i.e., mid- to high-nanomolar range) neoglycosides against the SKOV3 ovarian cancer line (see Figure 7). These data suggest the type of neoglycoside handle employed greatly influences desired activity. When taken into account that aglycons 9, 65, and 71 are of similar potency, the variation in activity between the methoxyamino-, hydroxyamino-, and acyl hydrazine-based neoglycosides implicates the nature of the glycosidic bond to be the most significant contributor wherein a prevalence of the acyclic nitrone or imine conjugate led to a reduction in potency.

Figure 7.

Figure 7

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Summary of GI50 data from the high-throughput growth inhibition assay of 66–70 hydroxyamines and 72–75 hydrazides (reciprocal values displayed). Comparisons were performed against methoxyamines 39 and 60, aglycons 65 and 71, and chlorambucil (1). GI50 data and error values are provided in the Supporting Information, Table 5. Representative cancer cell lines tested include: NCI-H460 (lung), A549 (lung), Du145 (prostate), SKOV3 (ovary), Hep3b (liver), SF268 (brain), MCF7 (breast), HT29 (colorectal), HCT15 (colorectal), H1299 (lung).

Conclusions

In summary, this study revealed a facile four step process, which could be conducted on gram scale in less than two hours of reaction time and required only a single chromatographic separation, to modify the drug chlorambucil (1) for chemoselective glycosylation. While prior syntheses of 1-glycoconjugates focused upon the use of typical metabolic sugars designed to enhance sugar-mediated uptake and led to modest overall improvements compared to 1,39,40 the current study revealed anticancer potency optimization was best accomplished via conjugation with novel non-metabolic sugars – culminating in the discovery of D-threoside 60 as the most active chlorambucil glycoside reported to date. The discovery of 60 opens the door to a series of new questions relating to the precise mechanism(s) of improvement – including among the many possibilities: i) modulation of uptake (via novel targeting of known transporters and/or even raising the possibility of new sugar transport/receptor mediated-processes); ii) intracellular stabilization of the alkylating reagent (basically extending the intracellular T1/2); iii) enhancing the productive agent-DNA interactions (e.g., DNA affinity and/or specificity); and/or even iv) alternative targeting of the active species (e.g., RNA, proteins, and/or membrane targets). While the specific mechanism(s) remain to be elucidated, it is important to note that precedent does exists for 1 glucosylation to alter the mechanism of cellular uptake.34,42

In addition to the lead discovery aspect of this project, it is also important to note that the corresponding in-depth product distribution analysis among N-alkoxyamino-, N-acyl hydrazine- and N-hydroxyamino-based neoglycosylation reactions revealed sugar-dependent partitioning among open and closed-ring neoglycosides in the latter two cases. Thus, this cumulative study also sheds new light (and a potential note of caution) on an under-appreciated chemical variability of glycoconjugates generated via the N-acyl hydrazine- and N-hydroxyamine-based chemoselective glycosylation methods more commonly applied in glycobiology.18

Experimental Section

Materials and General Methods

Mass spectrometric data were obtained on either a Waters (Milford, MA) LCT time-of-flight spectrometer for electrospray ionization (ESI) or a Varian ProMALDI (Palo Alto, CA) Fourier-transform ion cyclotron resonance mass spectrometer (FTICR) equipped with a 7.0 Tesla actively-shielded superconducting magnet and a Nd-YAG laser. NMR spectra were obtained on a Varian UnityInova 500 MHz instrument (Palo Alto, CA) using 99.8% CDCl3 with 0.05% v/v TMS or 99.8% CD3OD in ampoules. 1H and 13C chemical shifts were referenced to TMS (for CDCl3) or nondeuterated solvent (for CD3OD). Multiplicities are indicated by s (singlet), d (doublet), t (triplet), q (quartet), qui (quintet), m (multiplet), and br (broad). Chemical shift assignments for anomeric mixtures, where possible, are noted as α or β with the atom responsible for the shift. 1H NMR characterization was supplemented with gCOSY for all neoglycoside library members as well as 13C and gHSQC for pilot reactions and alternate handle compounds. Tetrahydrofuran was dried using a column of activated alumina. All other solvents were used as provided by the supplier. Reagents were obtained from Aldrich or Sigma and were used as received. Flash chromatography was performed using 40–63 μm particle sized silica gel. Thin layer chromatography was performed on aluminum-backed, 254 nm UV-active plates with a silica gel particle size of 60 μm. Library purity was assessed by reverse phase HPLC using a Varian (Walnut Creek, CA) ProStar unit with a Phenomenex (Torrance, CA) Luna C18 4.6 × 250 mm column running a H2O/MeCN 90/10 to 10/90 gradient over 13 m, followed by a 5 m isocratic flow, at a rate of 1.0 mL/m., A254 detection. Purity of the neoglycosides and aglycons was assessed to be greater than 95%, unless specified otherwise (see Table S4, Supporting Information).

N,O-Dimethyl 4-(4-N',N'-bis(2-chloroethyl)amino)phenylbutanamide (6)

Chlorambucil (1, 516 mg, 1.70 mmol) was dissolved in CH2Cl2 (15 mL) and cooled to 0 °C before adding N,O-dimethylhydroxylamine HCl (181 mg, 1.86 mmol) and N-methylmorpholine (200 μL, 1.82 mmol). The coupling agent EDAC (347 mg, 1.81 mmol) was then added to the reaction slowly over 5 min to ensure dissolution. After 60 min, the reaction was quenched with 5% HCl (20 mL) and the acidic layer extracted with CH2Cl2 (20 mL). The organic layers were combined, washed with sat. aq. NaHCO3 (20 mL) and the basic layer extracted with CH2Cl2 (20 mL). After combining the organic layers, washing with brine (20 mL), and drying with Na2SO4, solvent removal yielded a colorless oil (571 mg, 97%, Rf = 0.23 EtOAc:Hex 1:2) that was used without further purification. 1H NMR (CDCl3, 500 MHz) δ 7.09 (d, J = 8.8 Hz, 2 H), 6.62 (d, J = 8.8 Hz, 2 H), 3.71–3.65 (m, 4 H), 3.64 (s, 3 H), 3.62–3.59 (m, 4 H), 3.17 (s, 3 H), 2.58 (t, J = 7.7 Hz, 2 H), 2.44 (t, J = 7.0 Hz, 2 H), 1.95–1.88 (m, 2 H); 13C NMR (CDCl3, 125 MHz) δ 144.37, 131.17, 129.80, 112.31, 61.30, 53.74, 40.69, 34.33, 32.29, 31.35, 26.44; HRMS (ESI) m/z for C16H25Cl2N2O2 ([M+H]+) 347.1295, calc. 347.1288.

4-(4-N',N'-bis(2-chloroethyl)amino)phenylbutanal (7)

Weinreb amide 6 (571 mg, 1.64 mmol) was dissolved in anhydrous THF (8 mL) under Ar and cooled to 0 °C. A suspension of lithium aluminum hydride in anhydrous THF (1.0 M, 1 mL) was added in one aliquot. After 5 min, the reaction was quenched with sat. aq. KHSO4 (5 mL) followed by deionized water (5 mL). Extraction of the aldehyde was performed with Et2O (2 × 15 mL) and the organic layer was dried with Na2SO4. Solvent evaporation provided a yellowish oil (334 mg, 70%, Rf = 0.65 EtOAc:Hex 1:2). 1H NMR (CDCl3, 500 MHz) δ 9.80 (s, 1 H), 7.13 (d, J = 8.8 Hz, 2 H), 6.70 (d, J = 8.8 Hz, 2 H), 3.77–3.74 (m, 4 H), 3.69–3.66 (m, 4 H), 2.63 (t, J = 7.6 Hz, 2 H), 2.49 (td, J = 7.3, 1.5 Hz, 2 H), 2.00–1.94 (m, 2 H); 13C NMR (CDCl3, 125 MHz) δ 202.42, 144.46, 130.37, 129.69, 112.23, 53.56, 43.16, 40.60, 33.91, 23.93; HRMS (MALDI) m/z for C14H20Cl2NO ([M+H]+) 288.09091, calc. 288.09165.

N-Methoxy-4-(4-N',N'-bis(2-chloroethyl)amino)phenylbutanimine (8)

Aldehyde 6 (334 mg, 1.16 mmol) was dissolved in absolute EtOH (10 mL) followed by the addition of MeONH2•HCl (247 mg, 2.95 mmol) and Et3N (400 μL, 2.87 mmol). After 35 min, the solvent was removed in vacuo, providing the crude product as a white crystalline solid. The material was suspended in EtOAc:Hex 1:7 (50 mL) and filtered through a silica gel plug, which was flushed with solvent (400 mL). The purified imine was provided as a colorless oil containing a mixture of E- and Z-isomers (308 mg, 84%, Rf A = 0.51 Rf B = 0.43 EtOAc:Hex 1:7). 1H NMR (CDCl3, 500 MHz) δ 7.36 (t, J = 6.2 Hz, 1 H), 7.05 (d, J = 8.8 Hz, 2 H), 6.61 (d, J = 8.8 Hz, 2 H), 3.80 (s, 3 H), 3.69–3.65 (m, 4 H), 3.61–3.57 (m, 4 H), 2.55 (t, J = 7.7 Hz, 2 H), 2.18 (q, J = 7.7 Hz, 2 H), 1.75 (qui, J = 7.7 Hz, 2 H); 13C NMR (CDCl3, 125 MHz) δ 150.58, 144.38, 130.87, 129.74, 112.28, 61.27, 53.67, 40.61, 34.14, 29.03, 28.72; HRMS (ESI) m/z for C15H23Cl2N2O ([M+H]+) 317.1187, calc. 317.1182.

N-Methoxy-4-(4-N',N'-bis(2-chloroethyl)amino)phenylbutylamine (9)

Imine 8 (308 mg, 0.971 mmol) was dissolved in absolute EtOH (15 mL), cooled to 0 °C, and the reducing agent BH3•Et3N (710 μL, 4.83 mmol) was added in one aliquot. Concentrated HCl:EtOH 1:1 (800 μL) was then dripped in slowly over 5 min. The reaction was quenched with sat. aq. NaHCO3 (5 mL), 5 min after the acid solution was completely added, resulting in a white slurry. The organic solvent was removed under reduced pressure then more NaHCO3 (5 mL) solution was added. The aqueous material was extracted with CH2Cl2 (3 × 20 mL) and dried with Na2SO4. The crude oil was purified by flash chromatography (SiO2, EtOAc:Hex 1:2) providing the aglycon as a colorless oil (200 mg, 65%, Rf = 0.37 EtOAc:Hex 1:2). 1H NMR (CDCl3, 500 MHz) δ 7.05 (d, J = 8.6 Hz, 2 H), 6.60 (d, J = 8.7 Hz, 2 H), 5.50 (s br, 1 H), 3.69–3.65 (m, 4 H), 3.61–3.57 (m, 4 H), 3.50 (s, 3 H), 2.91 (t, J = 7.1 Hz, 2 H), 2.53 (t, J = 7.5 Hz, 2 H), 1.61–1.58 (m, 2 H), 1.55–1.49 (m, 2 H); 13C NMR (CDCl3, 125 MHz) δ 144.20, 131.63, 129.62, 112.22, 61.80, 53.65, 51.80, 40.61, 34.66, 29.32, 26.98; HRMS (ESI) m/z for C15H25Cl2N2O ([M+H]+) 319.1334, calc. 319.1339.

N-Hydroxy-4-(4-N',N'-bis(2-chloroethyl)amino)phenylbutanimine (64)

Aldehyde 7 (442 mg, 1.53 mmol) was dissolved in absolute EtOH (10 mL) followed by the addition of HONH2•HCl (269 mg, 3.87 mmol) and Et3N (550 μL, 3.95 mmol). After 25 min, the solvent was removed in vacuo, providing the crude product as a white flaky solid. The material was purified by column chromatography (SiO2, EtOAc:Hex 1:2), which yielded the imine as a colorless mixture of E- and Z-isomers (455 mg, 98%, Rf A = 0.53 Rf B = 0.45 EtOAc:Hex 1:2). 1H NMR (CDCl3, 500 MHz) δ 7.29 (t, J = 6.3 Hz, 1 H), 7.06 (d, J = 8.8 Hz, 2 H), 6.61 (d, J = 8.8 Hz, 2 H), 3.68–3.65 (m, 4 H), 3.61–3.58 (m, 4 H), 2.55 (t, J = 7.7 Hz, 2 H), 2.15 (q, J = 7.7 Hz, 2 H), 1.75 (qui, J = 7.7 Hz, 2 H); 13C NMR (CDCl3, 125 MHz) δ 147.98, 144.45, 131.01, 129.69, 112.22, 53.76, 40.59, 33.23, 29.10, 28.73; HRMS (ESI) m/z for C14H21Cl2N2O ([M+H]+) 303.1019, calc. 303.1025.

N-Hydroxy-4-(4-N',N'-bis(2-chloroethyl)amino)phenylbutylamine (65)

Imine 64 (455 mg, 1.50 mmol) was dissolved in absolute EtOH (10 mL), cooled to 0 °C, and the reducing agent BH3•Et3N (1.1 mL, 7.5 mmol) was added in one aliquot. Concentrated HCl:EtOH 1:1 (1.26 μL) was then dripped in slowly over 5 min. The reaction was quenched with sat. aq. NaHCO3 (5 mL), 5 min after the acid solution was completely added, resulting in a white slurry. The organic solvent was removed under reduced pressure then more NaHCO3 (5 mL) solution was added. The aqueous material was extracted with CH2Cl2 (4 × 25 mL) and dried with Na2SO4. The crude oil was purified by column chromatography (SiO2, MeOH:CH2Cl2 2:98 to 5:95) providing the aglycon as a colorless oil (302 mg, 66%, Rf = 0.36 MeOH:CH2Cl2 5:95). 1H NMR (CDCl3, 500 MHz) δ 7.05 (d, J = 8.7 Hz, 2 H), 6.61 (d, J = 8.7 Hz, 2 H), 5.23 (s br, 2 H), 3.70–3.64 (m, 4 H), 3.63–3.57 (m, 4 H), 3.40 (s, 2 H), 2.58–2.51 (m, 2 H), 1.96–1.84 (m, 2 H), 1.67–1.55 (m, 2 H); 13C NMR (CDCl3, 125 MHz) δ 144.60, 131.08, 129.84, 112.47, 53.81, 50.43, 40.88, 34.58, 29.00, 27.16; HRMS (ESI) m/z for C14H23Cl2N2O ([M+H]+) 305.1175, calc. 305.1182.

4-(4-N',N'-bis(2-chloroethyl)amino)phenylbutanoic hydrazide (71)

Chlorambucil (1, 319 mg, 1.05 mmol) was dissolved in THF (10 mL), along with N-hydroxysuccinimide (132 mg, 1.15 mmol) and DIC (180 μL, 1.16 mmol). The reaction was warmed to 40 °C and stirred for 5.5 h. Hydrazine (40 μL, 1.27 mmol), pyridine (10 μL), and DMAP (5 mg, 0.04 mmol) were then introduced and the reaction proceeded for another 20 min. The solvent was removed in vacuo yielding a yellowish solid, which was suspended in MeOH:CH2Cl2 3:97 and purified by column chromatography (SiO2, MeOH:CH2Cl2 3:97). The hydrazide product was collected as an opaque oil that solidified at −20 °C to a white amorphous solid (285 mg, 85% Rf = 0.39 MeOH:CH2Cl2 5:95). 1H NMR (CDCl3, 500 MHz) δ 7.31 (s br, 1 H), 7.05 (d, J = 8.7 Hz, 2 H), 6.61 (d, J = 8.7 Hz, 2 H), 3.90 (s br, 2 H), 3.71–3.66 (m, 4 H), 3.63–3.58 (m, 4 H), 2.54 (t, J = 7.6 Hz, 2 H), 2.17–2.13 (m, 2 H), 1.95–1.89 (m, 2 H); 13C NMR (CDCl3, 125 MHz) δ 173.80, 144.46, 130.47, 129.72, 112.26, 53.62, 40.66, 40.88, 34.10, 33.77, 27.19; HRMS (ESI) m/z for C14H22Cl2N3O ([M+H]+) 318.1140, calc. 318.1134.

General procedure for neoglycoside library synthesis, purification, and characterization

Aglycons 9, 65, or 71 (typically 0.12–0.16 mmol) were added to 1 dram vials along with stir fleas and dissolved in MeOH such that the aglycon was at a concentration of 90–100 mM. Glacial acetic acid (1.5 eq.) was introduced, reducing sugars (2 eq.) were added, the vials capped, and the vessels placed on a heating block/stir plate to react at 40 °C for 3–48 h. The vial caps were removed and the solvent evaporated by Speedvac apparatus (55 °C, 3 h). Crude neoglycosides were suspended in CH2Cl2 (200 μL) using a vortex mixer then loaded onto 2000 mg silica gel solid phase extraction (SPE) columns (Alltech, Deerfield, IL) that were prewashed with MeOH/CH2Cl2 2/98. The SPEs were eluted using a vacuum manifold, collecting fractions with a volume of approximately 2 mL. After the initial two fractions were obtained, eluting any unreacted aglycon or relatively nonpolar material, the following step gradients were used: MeOH/CH2Cl2 5/95 for tetroses, pentoses, and substituted hexoses; MeOH/CH2Cl2 10/90 for hexoses; MeOH/CHCl3 15/85 for disaccharides; MeOH/CHCl3 20/80 for glycuronosides. For polyprotected saccharides, a gradient of EtOAc/Hex 1/6 to 1/5 was used. Typically, all neoglycoside was eluted by the tenth or eleventh fraction, leaving unreacted sugar on the column. The fractions containing pure product were identified by TLC using UV light (254 nm) and p-anisaldehyde stain, then combined and dried. Library members were characterized by 1H and gCOSY NMR as well as either high-resolution ESI or MALDI mass spectrometry (see Tables S2 and S3, Supporting Information). Anomeric ratios were obtained by comparison of anomeric proton integration (see Tables S2 and S3, Supporting Information).

N-Methoxy-4-(4-N',N'-bis(2-chloroethyl)amino)phenylbutylamino-D-riboside (53)

Aglycon 9 (40 mg, 0.13 mmol) was placed into a 1 dram vial, dissolved in MeOH (1.34 mL), and mixed with glacial acetic acid (10.7 μL). After adding D-ribose (57 mg, 0.38 mmol), the reaction was capped, warmed to 40 °C and allowed to stir for 3 h. Solvent was subsequently removed in vacuo and the resulting crude solid suspended in MeOH:CH2Cl2 2:98 (250 μL) by vortex mixer. The mixture was purified by SPE (SiO2, MeOH:CH2Cl2 2:98 to 5:95), providing the white solid neoglycoside as a mixture of anomers (31 mg, 54%, Rf = 0.34 MeOH:CH2Cl2 5:95; α:β 1:3). 1H NMR (CD3OD, 500 MHz) δ 7.08 (d, J = 8.7 Hz, 2 H), 6.68 (d, J = 8.7 Hz, 2 H), 4.56 (d, J = 3.4 Hz, 0.25 H, α-H1'), 4.27 (d, J = 8.7 Hz, 0.75 H, β-H1'), 4.15 (dd, J = 5.4, 3.5 Hz, 0.25 H, α-H2'), 4.12 (s br, 0.75 H, β-H3'), 3.99 (t, J = 5.6 Hz, 0.25 H, α-H3'), 3.90–3.86 (m, 0.25, α-H4'), 3.74–3.68 (m, 5 H, α-H5A' + β-H5A'), 3.68–3.60 (m, 6.5 H, α-H5B' + β-H2' + β-H4' + β-H5B'), 3.58 (s, 3 H), 3.05–2.98 (m, 0.75 H, β-H2A), 2.97–2.92 (m, 0.25 H, α-H2A), 2.86–2.77 (m, 1 H, H2B), 2.56 (t, J = 7.2 Hz, 2 H), 1.71–1.56 (m, 4 H); 13C NMR (CD3OD, 125 MHz) δ 145.86, 132.73, 130.66, 113.61, 100.76 (α-C1'), 91.14 (β-C1'), 84.50 (α-C4'), 73.16 (α-C2'), 72.52 (α-C3'), 72.46 (β-C3'), 68.94 (β-C2'), 68.65 (β-C4'), 65.94 (β-C5'), 64.21 (α-C5'), 62.82, 54.76 (α-C2), 54.68, 54.05 (β-C2), 41.82, 35.76, 30.70, 28.12; HRMS (ESI) m/z for C20H32Cl2N2NaO5 ([M+Na]+) 473.1581, calc. 473.1587.

N-Methoxy-4-(4-N',N'-bis(2-chloroethyl)amino)phenylbutylamino-L-riboside (54)

Using the same procedure as 53, 9 (52 mg, 0.16 mmol) combined with L-ribose (48 mg, 0.32 mmol), yielded the anomeric mixture as a white solid (40 mg, 54%, Rf = 0.34 MeOH:CH2Cl2 5:95; α:β 1:3). 1H NMR (CD3OD, 500 MHz) δ 7.05 (d, J = 8.6 Hz, 2 H), 6.67 (d, J = 8.6 Hz, 2 H), 4.52 (d, J = 3.4 Hz, 0.25 H, α-H1'), 4.25 (d, J = 8.7 Hz, 0.75 H, β-H1'), 4.11 (dd, J = 5.4, 3.5 Hz, 0.25 H, α-H2'), 4.08 (s br, 0.75 H, β-H3'), 3.95 (t, J = 5.5 Hz, 0.25 H, α-H3'), 3.87–3.83 (m, 0.25, α-H4'), 3.71–3.67 (m, 5 H, α-H5A' + β-H5A'), 3.65–3.61 (m, 5.75 H, α-H5B' + β-H4' + β-H5B'), 3.60–3.59 (m, 0.75 H, β-H2'), 3.55 (s, 3 H), 3.00–2.95 (m, 0.75 H, β-H2A), 2.94–2.89 (m, 0.25 H, α-H2A), 2.81–2.76 (m, 1 H, H2B), 2.53 (t, J = 7.5 Hz, 2 H), 1.66–1.55 (m, 4 H); (CD3OD, 125 MHz) δ 145.96, 132.83, 130.69, 113.70, 100.85 (α-C1'), 91.20 (β-C1'), 84.59 (α-C4'), 73.24 (α-C2'), 72.61 (α-C3'), 72.56 (β-C3'), 69.02 (β-C2'), 68.74 (β-C4'), 66.00 (β-C5'), 64.28 (α-C5'), 62.81, 54.78 (α-C2), 54.76, 54.07 (β-C2), 41.86, 35.80, 30.75, 28.15; HRMS (ESI) m/z for C20H32Cl2N2NaO5 ([M+Na]+) 473.1579, calc. 473.1581.

N-Hydroxy-4-(4-N',N'-bis(2-chloroethyl)amino)phenylbutylamino-D-fucoside (66)

Using aglycon 65 (63 mg, 0.21 mmol), the mixture of compounds was yielded as a white solid (54 mg, 58%, Rf = 0.11 MeOH:CH2Cl2 5:95; α:β:nitrone 2.7:1:1.8). 1H NMR (CD3OD, 500 MHz) δ 7.25 (d, J = 5.8 Hz, 0.33 H, nitrone-H1'), 7.10–7.07 (m, 2 H), 6.70–6.67 (m, 2 H), 5.06 (dd, J = 5.8, 2.1 Hz, 0.33 H, nitrone-H2'), 4.40 (d, J = 4.9 Hz, 0.18 H, α-H1'), 4.30 (dd, J = 5.8, 4.9 Hz, 0.18 H, α-H2'), 4.08 (dd, J = 6.6, 1.8 Hz, 0.33 H, nitrone-H5'), 3.96 (dd, J = 7.5, 5.8 Hz, 0.18 H, α-H3'), 3.89 (dd, J = 8.7, 2.1 Hz, 0.33 H, nitrone-H3'), 3.86–3.79 (m, 1.33 H, nitrone-H2 + α-H5' + β-H1'), 3.73–3.70 (m, 4.49 H, β-H2'), 3.67–3.63 (m, 4.18 H, α-H4'), 3.61–3.58 (m, 0.98 H, β-H4' + β-H5'), 3.53–3.50 (m, 0.49 H, β-H3'), 3.48 (dd, J = 8.7, 1.8 Hz, 0.33 H, nitrone-H4'), 3.11–3.06 (m, 0.49 H, β-H2A), 3.05–3.01 (m, 0.18 H, α-H2A), 2.79–2.75 (m, 0.49 H, β-H2B), 2.74–2.68 (m, 0.18 H, α-H2B), 2.60–2.54 (m, 2 H), 1.91–1.87 (m, 0.66 H, nitrone-H3), 1.68–1.63 (m, 3.34 H), 1.30–1.26 (m, 3 H, H6'); 13C NMR (CD3OD, 125 MHz) δ 145.54 (nitrone-C1'), 144.64, 131.68, 129.45, 112.41, 99.46 (α-C1'), 94.89 (β-C1'), 86.70 (β-C2'), 78.52 (α-C2'), 77.32 (α-C3'), 74.99 (β-C3'), 73.68 (nitrone-C4'), 72.48 (β-C5'), 72.36 (β-C4'), 71.32 (nitrone-C3'), 67.76 (α-C5'), 67.38 (α-C4'), 67.26 (nitrone-C2'), 66.21 (nitrone-C5'), 64.69 (nitrone-C2), 54.87 (β-C2), 53.72 (α-C2), 53.49, 40.64, 34.59, 29.36, 28.16 (α/β-C3), 26.97 (nitrone-C3), 18.95 (α/β-C6'), 18.66 (nitrone-C6'); HRMS (MALDI) m/z for C20H32Cl2N2NaO5 ([M+Na]+) 473.15911, calc. 473.15805.

N-Hydroxy-4-(4-N',N'-bis(2-chloroethyl)amino)phenylbutylamino-D-glucurono-6,3-lactonide (67)

Using aglycon 65 (58 mg, 0.19 mmol), the mixture of compounds was yielded as a colorless syrup (49 mg, 56%, Rf = 0.28 MeOH:CH2Cl2 5:95; α:β:nitrone 0:1:2.5). 1H NMR (CD3OD, 500 MHz) δ 7.25 (d, J = 6.3 Hz, 0.71 H, nitrone-H1'), 7.10 (d, J = 8.6 Hz, 2 H), 6.70 (d, J = 8.6 Hz, 2 H), 5.11–5.08 (m, 0.71 H, nitrone-H2'), 4.85–4.83 (m, 0.29 H, β-H4'), 4.79 (d, J = 4.4 Hz, 0.29 H, β-H3'), 4.57 (d, J = 2.3 Hz, 0.29 H, β-H1'), 4.54 (d, J = 4.6 Hz, 0.71 H, nitrone-H5'), 4.49 (d, J = 2.0 Hz, 0.29 H, β-H5'), 4.47 (d, J = 2.8 Hz, 0.71 H, nitrone-H3'), 4.46 (d, J = 2.3 Hz, 0.29 H, β-H2'), 4.33 (dd, J = 4.6, 2.8 Hz, 0.71 H, nitrone-H4'), 3.91 (q, J = 7.0 Hz, 1.42 H, nitrone-H2), 3.74–3.71 (m, 4 H), 3.67–3.65 (m, 4 H), 2.99–2.96 (m, 0.29 H, β-H2A), 2.69–2.64 (m, 0.29 H, β-H2B), 2.59 (t, J = 7.5 Hz, 1.42 H, nitrone-H5), 2.69–2.64 (dd, J = 7.2, 6.7 Hz, 0.58 H, β-H5), 1.94–1.87 (m, 1.42 H, nitrone-H3), 1.68–1.62 (m, 2.58 H, β-H3 + H4); 13C NMR (CD3OD, 125 MHz) δ 176.39 (C6'), 144.87, 141.51 (nitrone-C1'), 130.76, 129.48, 112.45, 104.94 (β-C1'), 85.74 (β-C3'), 80.64 (nitrone-C3'), 77.57 (β-C4'), 77.24 (β-C2'), 70.90 (nitrone-C5'), 69.97 (nitrone-C4'), 69.65 (β-C5'), 66.13 (nitrone-C2'), 65.02 (nitrone-C2), 55.41 (β-C2), 53.42, 40.66, 34.55 (β-C5), 33.99 (nitrone-C5), 29.15 (β-C3), 28.17, 26.79 (nitrone-C3); HRMS (MALDI) m/z for C20H28Cl2N2NaO6 ([M+Na]+) 485.12254, calc. 485.12166.

N-Hydroxy-4-(4-N',N'-bis(2-chloroethyl)amino)phenylbutylamino-D-riboside (68)

Using aglycon 65 (50 mg, 0.16 mmol), the product mixture was yielded as a white solid that was visualized as a single spot by TLC (18 mg, 25%, Rf = 0.39 MeOH:CH2Cl2 10:90; α:β:nitrone 1:1:2). 1H NMR (CD3OD, 500 MHz) δ 7.17 (d, J = 6.3 Hz, 0.5 H, nitrone-H1'), 7.07 (d, J = 8.7 Hz, 2 H), 6.66 (d, J = 8.7 Hz, 2 H), 4.99 (dd, J = 6.3, 3.5 Hz, 0.5 H, nitrone-H2'), 4.51 (d, J = 2.9 Hz, 0.25 H, α-H1'), 4.18–4.17 (m, 0.25 H, α-H2'), 4.16 (d, J = 8.7 Hz, 0.25 H, β-H1'), 4.12–4.09 (m, 0.5 H, α/β-H4' + β-H2'), 3.89–3.86 (m, 0.5 H, α-H3' + α/β-H5A'), 3.86–3.85 (m, 1 H, nitrone-H2), 3.84–3.81 (m, 0.25 H, α/β-H5A'), 3.78 (dd, J = 11.5, 3.5 Hz, 0.5 H, nitrone-H3'), 3.74–3.67 (m, 5 H, nitrone-H5A' + α/β-H4' + α/β-H5B'), 3.65–3.58 (m, 5 H, nitrone-H5B' + α/β-H5B' + β-H3'), 3.55–3.51 (m, 0.5 H, nitrone-H4'), 3.08–3.02 (m, 0.25 H, α-H2A), 2.98–2.93 (m, 0.25 H, α-H2B), 2.74–2.67 (m, 0.5 H, β-H2), 2.57 (t, J = 7.4 Hz, 2 H), 1.92–1.86 (m, 2 H), 1.68–1.60 (m, 2 H); 13C NMR (CD3OD, 125 MHz) δ 144.79, 144.63 (nitrone-C1'), 130.80, 129.52, 112.45, 100.86 (α-C1'), 91.12 (β-C1'), 83.43 (α-C3'), 74.19 (nitrone-C3'), 72.98 (α-C2'), 72.31 (nitrone-C4'), 70.79 (β-C2'), 70.71 (α/β-C4'), 68.16 (nitrone-C2'), 67.88 (β-C3'), 67.55 (α/β-C4'), 64.85 (α/β-C5'), 64.82, 63.58 (nitrone-C5'), 62.59 (α/β-C5'), 53.54, 40.57, 34.09, 28.22, 26.78; HRMS (ESI) m/z for C19H30Cl2N2NaO5 ([M+Na]+) 459.1441, calc. 459.1424.

N-Acetoxy-4-(4-N',N'-bis(2-chloroethyl)amino)phenylbutylamino-D-riboside peracetate (69)

The D-riboside mixture designated 68 (23 mg, 0.053 mmol) was peracetylated by dissolving in THF (5 mL) and adding acetic anhydride (0.5 mL, 5 mmol), DMAP (1 mg, 0.008 mmol), and Et3N (0.5 mL, 4 mmol). After 20 min, the solvent was removed in vacuo and the residue was purified by SPE chromatograpy (SiO2, EtOAc:Hex 2:3). The product was collected as a mixture of anomers of a colorless oil (32 mg, >99%, Rf β = 0.38 Rf α = 0.32 EtOAc:Hex 2:3; α:β = 2:1). 1H NMR (CDCl3, 500 MHz) δ 7.06–7.04 (m, 2 H), 6.61 (d, J = 8.6 Hz, 2 H), 5.62 (s, 0.33 H, β-H3'), 5.27–5.25 (m, 0.67 H, α-H2'), 5.21 (dd, J = 6.1, 5.4 Hz, 0.67 H, α-H3'), 5.07 (dd, J = 9.2, 2.6 Hz, 0.33 H, β-H2'), 5.04–5.01 (m, 0.33 H, β-H4'), 4.89 (d, J = 2.9 Hz, 0.67 H, α-H1'), 4.58 (d, J = 9.2 Hz, 0.33 H, β-H1'), 4.33–4.31 (m, 0.67 H, α-H5A'), 4.23–4.20 (m, 0.67 H, α-H4'), 4.16 (dd, J = 11.7, 5.4 Hz, 0.67 H, α-H5B'), 3.97 (dd, J = 10.9, 5.4 Hz, 0.33 H, β-H5A'), 3.71–3.67 (m, 4.33 H, β-H5B'), 3.63–3.60 (m, 4 H), 3.20–3.15 (m, 0.33 H, β-H2A), 3.11–3.06 (m, 0.67 H, α-H2A), 2.99–2.91 (m, 1 H, α-H2B + β-H2B), 2.57–2.47 (m, 2 H), 2.11 (s, 3 H), 2.10 (s, 3 H), 2.09 (s, 3 H), 2.05 (s, 3 H), 1.67–1.46 (m, 4 H); 13C NMR (CDCl3, 125 MHz) δ 170.72, 169.76, 169.51, 169.49, 144.35, 131.49, 129.74, 112.36, 96.56 (α-C1'), 88.60 (β-C1'), 77.85 (α-C4'), 71.99 (α-C2'), 70.58 (α-C3'), 68.93 (β-C3'), 66.47 (β-C4'), 65.72 (β-C2'), 63.55 (α-C5'), 63.21 (β-C5'), 54.06, 53.79, 40.71, 34.55, 29.20, 26.53, 20.93, 20.88, 20.68, 20.65; HRMS (ESI) m/z for C27H38Cl2N2NAO9 ([M+Na]+) 627.1859, calc. 627.1847.

N-Hydroxy-4-(4-N',N'-bis(2-chloroethyl)amino)phenylbutylamino-D-threoside nitrone (70)

Using aglycon 65 (35 mg, 0.11 mmol), the neoglycoside nitrone was yielded as a colorless syrup (23 mg, 49%, Rf = 0.11 MeOH:CH2Cl2 5:95). 1H NMR (CD3OD, 500 MHz) δ 7.22 (d, J = 5.8 Hz, 1 H, H1'), 7.10 (d, J = 8.6 Hz, 2 H), 6.70 (d, J = 8.6 Hz, 2 H), 4.84 (dd, J = 5.8, 4.2 Hz, 1 H, H2'), 3.87–3.84 (m, 3 H, H3' + H4'), 3.75–3.72 (m, 4 H), 3.68–3.61 (m, 6 H), 2.59 (t, J = 7.4 Hz, 2 H), 1.93–1.87 (m, 2 H), 1.69–1.63 (m, 2 H); 13C NMR (CD3OD, 125 MHz) δ 146.11, 146.01 (C1'), 132.10, 130.72, 113.71, 73.93 (C3'), 68.77 (C2'), 65.95 (C4'), 64.09, 54.70, 41.85, 35.30, 29.41, 27.93; HRMS (MALDI) m/z for C18H28Cl2N2NaO4 ([M+Na]+) 429.13188, calc. 429.13183.

4-(4-N',N'-bis(2-chloroethyl)amino)phenylbutylhydrazido-βD-fucoside (72)

Using aglycon 71 (47 mg, 0.15 mmol), the product was yielded as a colorless syrup (46 mg, 67%, Rf = 0.28 MeOH:CH2Cl2 10:90). 1H NMR (CD3OD, 500 MHz) δ 7.09 (d, J = 8.6 Hz, 2 H), 6.70 (d, J = 8.6 Hz, 2 H), 3.86 (d, J = 8.7 Hz, 1 H, H1'), 3.74–3.72 (m, 4 H), 3.68–3.65 (m, 4 H), 3.64–3.62 (m, 2 H, H3' + H5'), 3.54 (dd, J = 9.5, 3.2 Hz, 1 H, H4'), 3.48 (d, J = 8.7 Hz, 1 H, H2'), 2.56 (t, J = 7.5 Hz, 2 H), 2.21 (t, J = 7.5 Hz, 2 H), 1.90 (qui, J = 7.5 Hz, 2 H), 1.28 (d, J = 6.4 Hz 3 H); 13C NMR (CD3OD, 125 MHz) δ 174.72, 144.86, 130.58, 129.53, 112.44, 91.30 (C1'), 74.29 (C4'), 72.12 (C3'), 72.03 (C5'), 68.38 (C2'), 53.45, 40.61, 34.02, 33.28, 27.65, 24.17, 15.93 (C6'); HRMS (MALDI) m/z for C20H31Cl2N3NaO5 ([M+Na]+) 486.15233, calc. 486.15330.

4-(4-N',N'-bis(2-chloroethyl)amino)phenylbutylhydrazido-D-glucurono-6,3-lactonide (73)

Using aglycon 71 (61 mg, 0.19 mmol), the mixture of compounds was yielded as a colorless syrup (42 mg, 44%, Rf A = 0.55 Rf B = 0.47 MeOH:CH2Cl2 10:90; α:β:imine = 0:1:1). 1H NMR (CD3OD, 500 MHz) δ 7.57 (d, J = 4.3 Hz, 0.35 H, imine A-H1'), 7.41 (d, J = 4.1 Hz, 0.15 H, imine B-H1'), 7.09 (d, J = 8.6 Hz, 2 H), 6.70 (d, J = 8.6 Hz, 2 H), 4.91 (dd, J = 6.4, 4.7 Hz, 0.5 H, β-H4'), 4.85–4.79 (m, 0.5 H, β-H3'), 4.78 (d, J = 1.4 Hz, 0.5 H, β-H1'), 4.68 (dd, J = 7.6, 4.3 Hz, 0.35 H, imine A-H2'), 4.63 (dd, J = 7.8, 4.1 Hz, 0.15 H, imine B-H2'), 4.57–4.54 (m, 1 H, imine-H4'/H5' + β-H2'), 4.53–4.50 (m, 1.35 H, imine A-H3' + imine-H4'/H5' + β-H5'), 4.46–4.44 (m, 0.15 H, imine B-H3'), 3.75–3.71 (m, 4 H), 3.68–3.65 (m, 4 H), 2.71–2.54 (m, 2 H), 2.27 (t, J = 7.5 Hz, 1 H), 2.20–2.15 (m, 1 H), 1.96–1.87 (m, 2 H); 13C NMR (CD3OD, 125 MHz) δ 176.71 (imine-C6'), 176.08 (β-C6'), 171.83, 148.17 (imine A-C1'), 144.93, 144.85 (imine B-C1'), 130.37, 129.50, 112.43, 99.79 (β-C1'), 84.14 (β-C3'), 81.62 (imine B-C3'), 81.53 (imine A-C3'), 77.83 (β-C4'), 70.96 (imine B-C4'/C5'), 70.88 (β-C2'), 70.11 (imine AC-4'/C5'), 69.97 (imine B-C4'/C5'), 69.84 (imine A-C4'/C5'), 69.49 (β-C5'), 69.20 (imine A-C2'), 69.59 (imine B-C2'), 53.40, 40.62, 33.99, 33.58 (β-C2), 33.47 (imine-C2), 27.36; HRMS (MALDI) m/z for C20H27Cl2N3NaO6 ([M+Na]+) 498.11631, calc. 498.11691

4-(4-N',N'-bis(2-chloroethyl)amino)phenylbutylhydrazido-D-threoside imine (74)

Using aglycon 71 (36 mg, 0.11 mmol), the neoglycoside imine was yielded as a colorless syrup (41 mg, 85%, Rf = 0.19 MeOH:CH2Cl2 10:90). 1H NMR (CD3OD, 500 MHz) δ 7.53 (d, J = 4.9 Hz, 0.6 H, imine A-H1'), 7.37 (d, J = 5.5 Hz, 0.4 H, imine B-H1'), 7.08 (d, J = 8.6 Hz, 2 H), 6.70 (d, J = 8.6 Hz, 2 H), 4.32 (t, J = 4.4 Hz, 0.6 H, imine A-H2'), 4.25 (m, 0.4 H, imine B-H2'), 3.78–3.70 (m, 5 H, imine-H3'), 3.69–3.60 (m, 6 H, imine-H4'), 2.65 (t, J = 7.5 Hz, 0.8 H, imine B-H2), 2.58 (t, J = 7.5 Hz, 1.2 H, imine A-H2), 2.26 (t, J = 7.5 Hz, 1.2 H, imine A-H4), 1.98–1.90 (m, 2.8 H, imine-H3 + imine B-H3); 13C NMR (CD3OD, 125 MHz) δ 171.67, 151.04 (imine A-C1'), 147.62 (imine B-C1'), 144.92, 130.40, 129.49, 112.44, 73.65 (imine B-C3'), 73.46 (imine A-C3'), 71.49 (imine B-C2'), 71.40 (imine A-C2'), 62.60 (imine-C4'), 53.42, 40.59, 34.19 (imine B-C2), 34.00 (imine A-C2), 33.56 (imine A-C4), 31.76 (imine B-C4), 27.41 (imine A-C3), 26.82 (imine B-C3); HRMS (MALDI) m/z for C18H27Cl2N3NaO4 ([M+Na]+) 442.12721, calc. 442.12708.

4-(4-N', N'-bis(2-chloroethyl)amino)phenylbutylhydrazido-D-xyloside (75)

Using aglycon 71 (60 mg, 0.19 mmol), the mixture of compounds was yielded as a colorless syrup (40 mg, 45%, Rf A = 0.36 Rf B = 0.29 MeOH:CH2Cl2 10:90; α:β:imine = 0:2.3:1). 1H NMR (CD3OD, 500 MHz) δ 7.54 (d, J = 5.0 Hz, 0.15 H, imine A-H1'), 7.38 (d, J = 5.5 Hz, 0.15 H, imine B-H1'), 7.09 (d, J = 8.6 Hz, 2 H), 6.70 (d, J = 8.6 Hz, 2 H), 4.45 (d, J = 3.8 Hz, 0.15 H, imine A-H2'), 4.40 (t, J = 4.9 Hz, 0.15 H, imine B-H2'), 3.89 (dd, J = 11.3, 5.4 Hz, 0.7 H, β-H5A'), 3.85 (d, J = 8.7 Hz, 0.7 H, β-H1'), 3.75–3.72 (m, 4.3 H, imine B-H3' + imine A/B-H4'), 3.68–3.65 (m, 4.75 H, imine A/B-H4' + imine-H5'), 3.59 (dd, J = 7.8, 4.0 Hz, 0.15 H, imine A-H3'), 3.52–3.45 (m, 0.7 H, β-H4'), 3.39–3.36 (m, 0.7 H, β-H3'), 3.23–3.16 (m, 1.4 H, β-H2' + β-H5B'), 2.56 (t, J = 7.5 Hz, 2 H), 2.27–2.24 (m, 0.6 H, imine-H2), 2.21–2.17 (m, 1.4 H, β-H2), 1.96–1.87 (m, 2 H); 13C NMR (CD3OD, 125 MHz) δ 174.67, 144.87, 130.53, 129.49, 112.43, 91.78 (β-C1'), 87.51 (imine A-C2'), 77.17 (β-C3'), 72.44 (imine A/B-C4'), 72.43 (imine B-C3'), 71.57 (imine A/B-C4'), 72.00 (imine B-C2'), 71.34 (β-C2'), 70.92 (imine A-C3'), 70.05 (β-C4'), 67.28 (β-C5'), 62.95 (imine-C5'), 53.43, 40.60, 33.99, 33.22, 27.67; HRMS (MALDI) m/z for C19H29Cl2N3NaO5 ([M+Na]+) 472.13712, calc. 472.13765.

Cell proliferation assays

Testing was performed by the Keck-UWCCC Small Molecule Screening Facility (Madison, WI). General carcinoma cell line maintenance, compound handling and assay protocols have been previously reported.20 Briefly, cells were plated in 50 μL volumes at a density of 500 cells per well in 384-well clear bottom tissue culture plates. Serial dilutions of 30 mM DMSO compound stock solutions were accomplished in 96-well plates using a BioTek Precision XS liquid handler (Winooski, VT) to a concentration 100× greater than that of the most dilute assay. Final dilutions were performed in a 384-well plate in quadruplicate using a Beckman-Coulter Biomek FX liquid handler with a 384 channel pipetting head (Fullerton, CA) and were stored at −20 °C when not in use. Compounds were then added to the culture plates by the Biomek FX handler and were incubated at 37 °C for 7 d in an atmosphere containing 5% CO2. The calcein AM reagent (acetoxymethyl ester; 10 μM) and ETHD-1 (100 μM; 30 μL total) were added, the treated cells incubated for 30 m at 37 °C, and plates read for fluorescent emission using a Tecan Safire2 microplate reader (Duram, NC) at the appropriate wavelengths. Cell titer-glo reagent (15 μL; Promega Corp., Madison, WI) was added and the plates incubated for 10 m at room temperature with gentle agitation to lyse the cells. Each plate was re-examined for luminescence to verify inhibition. GI50 values for cytotoxicity were determined using XLfit 4.2 as previously reported.20 For compound 60, the most active neoglycoside, the NCI60 human tumor cancer cell line screen was performed by the Developmental Therapeutics Program of the National Cancer Institute (Rockville, MD) as previously described.61,62

Supplementary Material

1_si_001

Acknowledgment

This work was supported by funding from the NIH (U19 CA113297 and AI52218) and the Laura and Edward Kremers Chair in Natural Products Chemistry (J.S.T.). Analytical support was provided by the School of Pharmacy Analytical Instrumentation Center and cancer cell line cytotoxicity screening was provided by the Keck UWCCC Small Molecule Screening Facility.

Footnotes

a

Abbreviations: BH3•Et3N, borane-triethylamine complex; CLL, chronic lymphocytic leukemia; CNS, central nervous system; DIC, N,N'-diisopropylcarbodiimide; DMAP, 4-(N',N'-dimethylamino)pyridine; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; GI50, growth inhibitory concentration for 50% of the cell population under study; GLUT1, glucose transporter 1; LAH, lithium aluminum hydride; NMM, N-methylmorpholine; SAAT1, sodium/amino acid transporter 1; SGLT3, sodium/glucose transporter 3; SPE, solid phase extraction; THF, tetrahydrofuran.

Supporting Information Available Experimental procedures and characterization data for aglycons 9, 65, and 71; characterization, figures, and growth inhibition data for the neoglycoside library; NCI60 anticancer screen data for 60; and NMR spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

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Supplementary Materials

1_si_001
NIHMS248350-supplement-1_si_001.pdf (1.8MB, pdf)

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