Alkyl-Substituted Polyaminohydroxamic Acids: A Novel Class of Targeted Histone Deacetylase Inhibitors

Abstract

The reversible acetylation of histones is critical for regulation of eukaryotic gene expression. The histone deacetylase inhibitors trichostatin (TSA, 1), MS-275 (2) and suberoylanilide hydroxamic acid (SAHA, 3) arrest growth in transformed cells and in human tumor xenografts. However, 1–3 suffer from lack of specificity among the various HDAC isoforms, prompting us to design and synthesize polyaminohydroxamic acid (PAHA) derivatives 6–21. We felt that PAHAs would be selectively directed to chromatin and associated histones by the positively charged polyamine side chain. At 1 μM, compounds 12, 15 and 20 inhibited HDAC by 74.86, 59.99 and 73.85%, respectively. Although 20 was a less potent HDAC inhibitor than 1, it was more potent than 2, more effective as an initiator of histone hyperacetylation, and significantly more effective than 2 at re-expressing p21^Waf1 in ML-1 leukemia cells. On the basis of these results, PAHAs 6–21 represent an important new chemical class of HDAC inhibitors.

Introduction

The reversible acetylation of histones, mediated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), plays a critical role in chromatin architecture and hence in regulation of gene expression.^1,2 Acetylation of cationic lysine tails in nucleosome-associated histones neutralizes charge and promotes relaxation of chromatin, leading to transcriptional activation. Conversely, deacetylation of these lysine residues promotes formation of condensed chromatin, and transcription is repressed. In some tumor cell types, excessive hypocetylation of histones results in the underexpression of growth regulatory factors such as the cyclin dependent kinase inhibitor p21^Waf1 and thus contributes to the development of cancer.^1,2 Histone hyperacetylation caused by HDAC inhibitors such as trichostatin (TSA, 1), MS-275 (2) and SAHA (3) (Figure 1) can cause growth arrest in a wide range of transformed cells and can inhibit the growth of human tumor xenografts.^1-4 Cyclic peptide HDAC inhibitors such as apicidin, depsipeptide, trapoxin and CHAPs have also shown promising activity in cell culture and in vivo. Current structure/activity studies involving analogues of 1–3 have focused largely on modifications to the aromatic ring moiety and the aliphatic linker region present in these molecules.⁴ Although they are effective both in vitro and in vivo, HDAC inhibitors typified by 1–3 suffer from lack of specificity among the various forms of HDAC, including deacetylases that target nonhistone proteins. Further, compounds 1 and 2 (and to some degree 3) produce side effects through activity in noncancerous cells. Thus, it would be desirable to identify potent HDAC inhibitors that restore the expression of normal tumor suppressor factors without producing significant dose-limiting toxicity.⁵ To address these problems, we designed and synthesized a series of polyaminohydroxamic acid (PAHA) derivatives that incorporate structural features of the polyamines spermidine and spermine (4 and 5, respectively, Figure 1) and the hydroxamic acid moiety commonly found in active HDAC inhibitor molecules such as 1 and 3. This strategy was developed based on the observation that polyamine analogues can exert antitumor effects by virtue of their high affinity for DNA.^6-9 We postulated that these compounds could enter cells using the polyamine cellular transport system,^6,10 and could be selectively directed to DNA and associated histones, by virtue of the positively charged polyamine portion of the structure. In addition, it has been shown that histone deacetylases differ in primary sequence at specific residues in the rim region outside the lysine binding site.³ Therefore, it may be possible to produce isoform specific inhibitors for individual HDACs by altering the polyamine chain composition and the terminal alkyl group on that chain. Using these design criteria, we successfully identified three lead compounds from a library consisting of only 16 analogues.

Figure 1.

Structures of trichostatin, MS-275, SAHA, spermidine and spermine.

Chemistry

Depending on structure, one of 5 synthetic routes was used to produce compounds 6–21, as outlined in Schemes 1–5. The structures and molecular weights of analogues 6–21 are summarized in Table 1. The synthesis of compounds 6, 8, 11, 13, 17 and 18 is shown in Scheme 1. Commercially available N-(3-bromobutyl)phthalimide 22 was first converted to the corresponding azide 23 (NaN₃, DMF),¹¹ which was immediately reduced to the corresponding amine 24 by catalytic hydrogenation (10% Pd/C, 25 psi).¹² The desired aralkyl group was then added by reductive amination¹³ of the appropriate aldehyde (NaCNBH₃, AcOH) to afford the phthalimide-protected diamines 25a–c. The secondary amine was then N-Boc protected (Boc)₂O, NaHCO₃, NaCl)¹⁴ to provide 26a–c, followed by removal of the phthalimide (methanolic NH₂NH₂)¹⁵ to give 27a–c. The free primary amine was then coupled to acid chloride 28a (n)1) or 28b (n) 3), yielding 29a–e.^13,16 The methyl ester in 29a–e was cleaved (1N LiOH),¹¹ resulting in the free acids 30a–e, and these intermediates were then converted to hydroxamic acids 31a–e in a two step process¹⁷ involving formation of an activated mixed anhydride (ethylchloroformate, THF) followed by addition of hydroxylamine (1.76 M NH₂OH in MeOH). Removal of the N-Boc protecting group (20% TFAA in CH₂Cl₂)¹⁴ then afforded compounds 6, 8, 13, 17 and 18 as trifuoroacetate salts. During the reductive amination step, one of the isolated products was tertiary amine 25d, which resulted from addition of two equivalents of 2-(phenyl)benzaldehyde. Elaboration of this intermediate as described above resulted in the formation of target analogue 11.

Scheme 1.

Scheme 5.

Table 1.

Comparison of the in Vitro Inhibitory Activity of 1, 2 and 6–20 at 1 μM Concentration Using an Isolated HDAC Assay Kit

Structure	No.	Empirical Formula	MW	HDAC activity remaining
	1	C17H22NO3	288.35	0% (control)
	2	C21H20N4O3	376.41	0% (control)
	6	C23H31N3O3	397.51	83.66
	7	C31H54N6O3Br4	1004.01	ND (insolb)
	8	C18H29N3O3	335.44	55.83
	9	C22H50N6O3Br4	766.29	ND (insolb)
	10	C26H58N6O3Br4	822.39	58.97
	11	C36H41N3O3	563.73	96.26
	12	C24H44N4O3Br2	596.44	25.14
	13	C23H37N4O5F3	508.56	66.11
	14	C24H43N3O3Br2	581.42	98.52
	15	C22H41N5O3Br2	583.40	40.01
	16	C26H47N3O3Br2	609.48	50.64
	17	C21H33N4O5F3	478.51	89.68
	18	C22H34N3O5F3	477.52	46.80
	19	C23H37N4O5F3	544.56	84.40
	20	C30H48N4O3Br2	672.51	26.15
	21	C28H44N4O3Br2	644.46	80.25

The synthetic route used to produce target analogues 12, 16, 20 and 22 is outlined in Scheme 2. The previously described dimesitylated phthalimide 32¹⁸ was monoalkylated with a suitable aralkyl halide (NaH, DMF)¹⁹ to produce 33a-b. Subsequent removal of the phthalimide (methanolic NH₂NH₂)¹⁵ provided the free amines 34a–b. Coupling with 28a or 28b as described above afforded intermediates 35a–d, followed by ester cleavage (1 N LiOH)¹¹ to give 36a–d. These intermediates were then converted to the corresponding hydroxamic acids 37a–d as described above,¹⁷ and removal of the mesityl protecting groups (30% HBr in AcOH)^20,21 afforded target compounds 12, 16, 20 and 21 as dihydrobromide salts. Direct deprotection of carboxylic acid 36a (30% HBr in AcOH)^20,21 produced target compound 14.

Scheme 2.

The synthesis of compounds 7, 9 and 10 is shown in Scheme 3. Tetramesitylnorspermine 38^18,19 was mono-alkylated (1.1 equiv of N-(3-bromobutyl)phthalimide 22, NaH, DMF)^18,19 to give 39, followed by a second alkylation²² with the appropriate alkyl- or aralky halide (NaH, DMF) to provide 40a–c. Removal of the phthalimide (methanolic NH₂NH₂)¹⁵ followed by coupling to acid chloride 28a as described above then yielded the fully protected intermediates 41a–c. Conversion of the ester in 41a–c was then accomplished in three steps as described above,^11,17 resulting in hydroxamates 42a–c. Deprotection (30% HBr in AcOH)^20,21 then afforded compounds 7, 9 and 10 as tetrahydrobromide salts.

Scheme 3.

The synthesis of compound 19 is outlined in Scheme 4. Phthalimide 24 was converted to intermediate 25c by reductive amination (4-dimethylaminobenzaldehyde, NaCNBH₃, AcOH),¹³ followed by coupling to acid chloride 28b as described above^13,16 to provide compound 43. Removal of the phthalimide (methanolic NH₂NH₂)¹⁵ followed by N-Boc protection¹⁴ afforded 44, and subsequent conversion of the ester to the corresponding hydroxamate as described above^11,17 produced 45. Removal of the N-Boc group (20% TFAA in in CH₂Cl₂)¹⁴ then afforded the desired 19 as the trifluoroacetate salt.

Scheme 4.

The synthesis of analogue 15 is described in Scheme 5. Removal of the phthalimide¹⁵ from compound 32 and reductive amination (4-dimethylaminobenzaldehyde, NaCNBH₃, AcOH)¹³ resulted in 46, which was then coupled to acid chloride 28a^13,16 to afford intermediate 47. The ester was converted to the corresponding hydroxamic acid 48 as described above,¹⁷ and then removal of the mesityl protecting groups (30% HBr in AcOH)^20,21 resulted in the formation of analogue 15 as the dihydrobromide salt.

Biological Evaluation

Compounds 6–21 were evaluated for their ability to inhibit isolated HDAC at 1 μM in a commercially available assay (Fluor de Lys Assay System, Biomol International LP, Plymouth Meeting, PA), employing 1 μM 1 and 2 as positive controls. The results of these studies are summarized in Table 1. Three of these analogues, compounds 12, 15 and 20, reduced HDAC activity by 74.86, 59.99 and 73.85%, respectively, and as such were selected for more extensive studies. These three compounds were subjected to a dose response analysis using the same commercial assay kit, and varying the concentration of inhibitor between 0.5 and 1000 nM, with 1 as a positive control. As shown in Figure 2, compounds 12, 15 and 20 were essentially equipotent over the concentration range tested (IC₅₀) = 400 nM). Compounds 12, 15 and 20 act as potent inhibitors of HDAC that are significantly less effective than 1, but more effective than 2⁴ in this isolated enzyme assay system.

Figure 2.

In vitro dose–response for inhibition of HDAC caused by trichostatin and PAHAs 12, 15 and 20. The enzyme preparation was exposed to a concentration range of each inhibitor as described in the Experimental section. Each data point is the average of three determinations that in each case differed by 3% or less.

The most potent inhibitor of isolated HDAC, compound 20, was next evaluated in a series of cell proliferation studies in the ML-1 human myelocytic leukemia cell line. The results of these studies are outlined in Figure 3. Compound 20, as well as the positive controls 1 and 2, were compared at concentrations between 0.1 and 100 μM, and cell viability was determined in the ML-1 cultured cell preparation at 3 and 7 days (Figure 3) by direct cell count. Significant toxicity was noted in the presence of 1 and 2, with IC₅₀ values less than 10 μM in all cases. By contrast, compound 20 produced significantly lower cell toxicity than 1 and 2, especially at concentrations greater than 10 μM. These effects were similar when cell viability was monitored at either day 3 or 7. Comparable results were obtained in a separate set of experiments where cell proliferation was measured by a standard MTT assay procedure (data not shown).

Figure 3.

Toxicity of 1, 2 and compound 20 to ML-1 myelocytic leukemia cells in culture. Cells were exposed to a range of concentrations of the inhibitor, and cell viability was determined by direct cell count. Panel A: 3 days of treatment; Panel B: 7 days of treatment. Each data point is the result of three separate determinations that in each case differed by 3% or less.

Compound 20 was also compared to the HDAC inhibitor 2 (IC₅₀) 4.8 μM)⁴ for the ability to promote hyperacetylation of histones H3 and H4 in the ML-1 cell line, as shown in Figure 4. At 1 μM, compound 20 produced higher levels of acetylated H3 and H4 than 2 after 24 h, as determined by Western blot analysis. Histone H2a levels were also determined as an internal standard, and the levels of this protein did not change in the presence of 2 or 20. Re-expression of the cyclin dependent kinase inhibitor p21^Waf1 was also determined in the presence of 2 and 20, and 20 was found to be significantly more effective at promoting the re-expression of this protein. Taken together, the data shown in Figures 2-4 suggest that 20 produces more dramatic effects on ML-1 histone acetylation than 2, even though it is only a marginally more potent HDAC inhibitor in the in vitro enzyme assay.

Figure 4.

Acetylation of histone H3 and H4 and expression of p21^WAF1/CIP1 in ML-1 cells. ML-1 cells were incubated for 24 h with compound 20 prior to Western blot analysis. Compound 2 was used as a positive control.

Discussion

Well characterized HDAC inhibitors such as 1–3 and related analogues typically contain three structural features that are thought to be required for optimal activity: an aromatic cap group, an aliphatic chain and a metal binding functional group (Figure 5). Based on molecular modeling studies involving the histone deacetylase-like protein (HDLP), these molecules are thought to bind in a pocket in the enzyme active site that includes a channel region flanked by a zinc ion on one end, and a region that binds the cap group on the other end.² In this model, the aromatic group and aliphatic chain of the inhibitor are buried in the enzyme pocket in such a way that the metal binding moiety coordinates the zinc ion that is required for activity. For these reasons, compounds such as 1–3 do not inhibit Class III HDACs, since they do not require zinc for activity. As was mentioned above, the development of additional HDAC inhibitors has focused on modifications to the aliphatic linker group and the aromatic moiety. Somewhat less attention has been paid to the metal binding group, which is typically a hydroxamic acid. We reasoned that this model could be expanded by the addition of a polyamine chain to the structural model for active HDAC inhibitors, as shown in Figure 5. It was hypothesized that the polyamine portion would impart beneficial effects on activity, including cellular import of the molecule by the polyamine transport system, improved nuclear localization of the analogue, enhanced affinity of compounds for chromatin, and the potential to interfere with the association of HDACs with histone proteins. These suppositions were in part supported by data showing that the polyamines spermidine and spermine are known to inhibit yeast HDAC,²³ although it is unclear whether this is a direct inhibition at the active site or some other effect such as inhibition of HDAC binding to chromatin. It has also been shown that the amino acid residues that interact with 1 in the binding region of HDLP are highly conserved in the individual HDAC isoforms. However, the amino acids in the surrounding rim area are less conserved.³ We further hypothesized that the polyamine tail shown in Figure 5 would be available to bind to amino acids in this rim area, and that variations in charge, carbon chain length and the nature of the R group could be used to develop isoform-specific inhibitors. Using these parameters for the design of novel HDAC inhibitors, we were able to successfully identify three lead compounds from a library of only 16 analogues. Preliminary biological data suggests that some of our suppositions could be true, but additional experiments now being conducted (transport studies, DNA binding analysis, cell cycle studies, determination of activity against individual HDAC isoforms and molecular modeling) will be necessary to demonstrate the precise mechanism of these analogues. Although 20 is more potent with respect to inducing p21^Waf1 re-expression, it demonstrates less overt growth inhibition in ML-1 cells in vitro than either 1 or 2. This feature may be therapeutically exploitable in that the re-expression of previously inactivated growth regulatory genes without overt cytotoxicity may restore more normal growth control to transformed cells, thus making them less tumorigenic and potentially more susceptible to combination treatment with other cytotoxic agents. This possibility is currently being examined experimentally.

Figure 5.

Refined structural design model for polyaminohydroxamate HDAC inhibitors.

Preliminary examination of the structure/activity correlations for compounds 6–21 reveals interesting trends that will be studied through the synthesis of additional analogues. It is of interest to note that of the three most active analogues (12, 15 and 20), only compound 15 has a traditional cap group. Compounds 12 and 20 do contain aromatic groups at the terminal nitrogens of their polyamine chains, but they are unlikely to be positioned in the aromatic binding region of HDAC due to their distance from the hydroxamic acid moiety. This indicates that the polyamine portion of the chain may in part be responsible for the activity of these analogues, since each molecule contains two nitrogens that are charged at physiological pH. Not surprisingly, conversion of the hydroxamic acid moiety in 12 to a carboxyl group (as in 14) essentially abolished inhibitory activity. When the linker region of 12 was expanded from four to six carbons to produce 16, inhibitory activity was reduced by nearly 35%. By contrast, compound 20, with a six-carbon linker region, was nearly 4 times as active as an HDAC inhibitor as 21, which has a four-carbon linker region. Compounds with either one or four protonated nitrogens in the polyamine segment were generally only moderately active. These SAR correlations are preliminary, and additional data from an expanded series of analogues is required to generate a more substantive pharmacophore. The synthesis of additional analogues and more extensive biological evaluation procedures are currently underway in our laboratories, and will be published in a subsequent manuscript.

Experimental Section

All reagents were purchased from Aldrich Chemical Co. (Milwaukee, WI), Sigma Chemical Co. or Acros Chemical (Chicago, IL) and were used without further purification except as noted below. Pyridine was dried by passing it through an aluminum oxide column and then stored over KOH. Triethylamine was distilled from potassium hydroxide and stored in a nitrogen atmosphere. Methanol was distilled from magnesium and iodine under a nitrogen atmosphere and stored over molecular sieves. Methylene chloride was distilled fron phosphorus pentoxide and chloroform was distilled from calcium sulfate. Tetrahydrofuran was purified by distillation from sodium and benzophenone. Dimethylformamide was dried by distillation from anhydrous calcium sulfate and was stored under nitrogen. Preparative scale chromatographic procedures were carried out using E. Merck silica gel 60, 230–440 mesh. Thin-layer chromatography was conducted on Merck precoated silica gel 60 F-254. Ion exchange chromatography was conducted on Dowex 1 × 8–200 anion-exchange resin. Compounds 32 (Scheme 2) and 38 (Scheme 3) were synthesized as previously described.

All ¹H and ¹³C NMR spectra were recorded on a General Electric QE-300 or a Varian Mercury 400 MHz spectrometer, and all chemical shifts are reported as δ values referenced to TMS or DSS. Infrared spectra were recorded on a Nicolet 5DXB FT-IR spectrophotometer and are referenced to polystyrene. In all cases, ¹H NMR, ¹³C NMR, and IR spectra were consistent with assigned structures. Melting points were recorded on a Thomas-Hoover Capillary melting point apparatus and are uncorrected. Mass spectra were recorded on a Kratos MS 80 RFA (EI and CI) or Kratos MS 50 TC (FAB) mass spectrometers. Microanalyses were performed by Galbraith Laboratories, Knoxville, TN, and were within 0.4% of calculated values.

N-[4-(Azido)butyl]phthalimide (23)

A 1.0 g portion of N-[4-(bromo)butyl]phthalimide 22 (0.0035 mol) was dissolved in 10 mL of DMF, and to this solution was added 0.290 g (0.0044 mol) of sodium azide. The reaction was then allowed to stir for 5 h under nitrogen, after which the reaction mixture was concentrated in vacuo to yield a white semisolid. The semisolid was dissolved in water and extracted with three 50 mL portions of ethyl acetate, the combined organic layers were dried over anhydrous magnesium sulfate and filtered, and the solvent was removed to afford 23 (0.760 g, 88%) as a white amorphous powder. This preparation was used in the next reaction without further purification. ¹H NMR (400 MHz CDCl₃) δ 1.6–1.68 (m, 2H), 1.74–1.82 (m, 2H), 3.3 (t, J = 7.2 Hz, 2H), 3.7 (t, J = 7.2 Hz, 2H), 7.71–7.73 (m, 2H), 7.83–7.86 (m, 2H). ¹³C NMR(400 MHz CDCl₃) δ 26.1, 28.6, 44.02, 53.6, 127.4, 128.0, 132.34, 134.12, 168.52, 171.0.

N-[4-(Amino)butyl]phthalimide (24)

Compound 23 (0.760 g, 0.0031 mol) was dissolved in 50 mL of ethanol along with 0.100 g of 10% Pd/C, and the suspension was hydrogenated at 25psi for 12 h. The reaction mixture was then filtered, and the filtrate was concentrated in vacuo to yield 24 as an amorphous white solid (0.620 g, 92%) that was of sufficient purity to use in the next reaction. ¹H NMR (400 MHz CD3OD) δ 1.6–1.8 (m, 4H), 2.9 (t, J = 7.2 Hz, 2H), 3.7 (t, J = 7.6 Hz, 2H), 7.9 (m, 4H). ¹³C NMR (400 MHz CDCl₃) δ 26.1, 33.8, 44.0, 46.4, 127.4, 128.0, 132.23, 134.12, 168.25, 171.0

N-{4-[(N,N-Dimethylamino)benzyl]butyl}phthalimide (25c)

A 0.589 g portion of 24 (0.0027 mol) was dissolved in 20 mL of dichloroethane, and to this solution N,N-dimethylaminobenzaldehyde (0.477 g, 0.0032 mol) was added along with 0.186 g (0.0031 mol) of acetic acid. The reaction was allowed to stir at room temperature for 20 min. after which time sodium cyanoborohydride (0.220 g, 0.0035 mol) was dissolved in 3 mL of methanol and added to the reaction, which was then allowed to stir for an additional 12 h. The reaction mixture was concentrated on a rotary evaporator, and the resulting yellow oil was dissolved in water and extracted with three 50 mL portions of chloroform. The organic layers were combined, washed with brine, and then dried over anhydrous magnesium sulfate. The solution was filtered and concentrated in vacuo to give crude 25a as a cloudy yellow oil. The crude compound was purified using column chromatography (hexane: ethyl acetate 1:3 followed by ethyl acetate:methanol 2:1), to yield 25a as a clear yellow oil (0.810 g, 85.4%). ¹H NMR (400 MHz CDCl₃) δ 1.55 (q, J = 7.2 Hz, 2H), 1.72 (q, J = 7.2 Hz, 2H), 2.65 (t, J = 7.6 2H), 2.92 (s, 6H), 3.69 (s, 2H), 3.71 (t, J =7.2 Hz,2H), 6.7 (d,2H), 7.1 (d, 2H),7.68-7.71 (m, 2H), 7.81–7.83 (m,2H). ¹³C NMR (400 MHz, CDCl₃) δ 24.35, 25.35, 40.9, 50.06, 112.85, 127.4, 128.0, 128.6, 132.34, 134.12, 150.0, 168.52, 169.0.

N-{4-[4-(Methyl)benzylamino]butyl}phthalimide (25b)

Compound 25b was synthesized from 24 and 4-methylbenzaldehyde according to the procedure used to synthesize 25c in 73.2% yield. ¹H NMR (400 MHz, CDCl₃) δ 1.53 (q, J = 7.2 Hz, 2H), 1.70 (q, J = 7.2 Hz, 2H), 2.31(s, 3H), 2.62 (t, J = 7.6 2H), 6H), 3.68 (s, 2H), 3.70 (t, J = 7.2 Hz,2H), 6.7 (d,2H), 7.1 (d, 2H),7.68–7.71 (m, 2H), 7.81–7.83 (m,2H). ¹³C NMR (400 MHz CDCl₃) δ 25.52, 26.19, 37.85, 46.02, 53.65, 60.61, 123.41, 127.36, 127.95, 129.33, 132.34, 134.12, 136.92, 168.59.

N-{4-[2-(Phenyl)benzylamino]butyl}phthalimide (25a)

Compound 25a was synthesized from 24 and 2-(phenyl)benzaldehyde according to the procedure used to synthesize 25c in 75% yield. ¹H NMR (400 MHz CDCl₃) δ 1.42 (q, J = 7.6 Hz, 2H), 1.59 (q, J = 7.2 Hz, 2H), 2.54 (t, J = 7.2 Hz, 2H), 3.59 (t, J = 7.2 Hz, 2H), 3.84 (s, 2H), 7.19–7.45 (complex m, 9H), 7.77–7.81 (m, 4H). ¹³C NMR (400 MHz CDCl₃) δ 25.52, 26.18, 37.8, 47.15, 48.07, 123.38, 126.98, 127.34, 127.86, 128.461,129.37, 139.06, 132.35, 134.10, 141.0, 146.9, 168.50

1-N-{4-[(N,N-Dimethyl)amino]benzyl}-1-N-[(tert-butyloxy)carbonyl]-4-phthalimidobutylamine (26c)

A 0.800 g portion of 25c (0.0023 mol) was dissolved in 20 mL of dichloromethane, and the reaction mixture was cooled to 0 °C. To this mixture was added an aqueous solution of sodium bicarbonate (0.220 g, 0.0027 mol) and sodium chloride (0.160 g, 0.0027 mol), and the reaction was allowed to stir at 0 °C for 30 min. Di-tert-butyl dicarbonate (0.596 g, 0.0027 mol) was dissolved in 5 mL of dichloromethane, and the solution was slowly added to the reaction. The mixture was allowed to stir at 0 °C for an additional 10 min and warmed to room temperature, followed by reflux for 12 h. The reaction mixture was cooled and extracted with three 25 mL portions of dichloromethane. The combined organic layers were washed with 50 mL of saturated sodium bicarbonate and 50 mL of saturated sodium chloride solution and then dried over anhydrous magnesium sulfate. The mixture was then filtered, and the solvent was removed in vacuo to yield crude 26c. The crude compound was purified on a silica gel column eluted with hexane:ethyl acetate (4:3) and then ethyl acetate (100%), to yield compound 26c as a clear yellow oil (0.950 g, 91.5%). ¹H NMR (400 MHz CDCl₃) δ 1.4 (s, 9H), 1.51–1.61 (broad m, 4H), 2.89 (s, 6H), 3.1–3.18 (broad m, 2H), 3.65 (t, 2H), 4.3 (s, 2H), 6.65 (d, J = 6.4 Hz, 2H), 7.09 (broad s, 2H), 7.68–7.71 (m, 2H), 7.81–7.83 (m, 2H). ¹³C NMR (400 MHz CDCl₃) δ 25.52, 26.19, 28.66, 37.85, 46.02, 53.65, 60.61, 79.82 123.41,127.36, 127.95, 129.33, 132.34,134.12, 136.92, 168.59. IR (cm⁻¹) 3410.2, 2942.8, 1651.8, 1555.8, 1532.2, 1460.12, 1105.32.

1-N-[4-(Methyl)benzyl]-1-N-[(tert-butyloxy)carbonyl]-4-phthalimidobutylamine (26b)

Compound 26b was synthesized from 25b according to the procedure used to synthesize 25c in 92% yield. ¹H NMR (400 MHz CDCl₃) δ 1.44 (s, 9H), 1.53–1.63 (m, 4H), 2.30 (s, 3H), 3.14–3.22 (broad d, 2H), 3.66 (t, J = 7.2 Hz, 2H), 4.36 (s, 2H), 7.09 (s, 2H), 7.71 (m, 2H), 7.84 (m, 2H). ¹³C NMR (400 MHz CDCl₃) δ 24.64, 26.19, 28.71, 39.03, 40.92, 51.77, 60.61, 79.82 112.85, 123.41,127.36, 127.95, 129.33, 132.34,134.12, 136.92, 150.66, 156.17. IR (cm⁻¹) 2962.5, 2942.8, 1768.7, 1710.3, 1684.3, 1619.4, 1487.2, 1365.4, 1301.4.

1-N-[2-(Phenyl)benzyl]-1-N-[(tert-butyloxy)carbonyl]-4-phthalimidobutylamine (26a)

Compound 26a was synthesized from 25a according to the procedure used to synthesize 25c in 90.2% yield. ¹H NMR (400 MHz CDCl₃) δ 1.38–1.42 (m, 11H), 1.52 (m, 2H), 2.96 (broad s 1H), 3.09 (broad s 1H), 3.56 (broad s, 2H), 4.34 (s, 1H), 4.43 (s, 1H), 7.18 (d, J = 7.6 Hz,1H), 7.21–7.4 (m,8H),7.70–7.77 (m, 2H), 7.8–7.83(m, 2H). ¹³C NMR (400 MHz CDCl₃) δ 25.52, 26.18, 27.63, 28.62, 37.8, 47.15, 48.07, 79.83, 85.4123.38, 126.98, 127.34, 127.86, 128.461,129.37, 139.06, 132.35, 134.10, 141.0, 146.9, 168.50.

1-N-{4-[(N,N-Dimethyl)amino]benzyl}-1-N-[(tert-butyloxy)carbonyl]-4-aminobutylamine (27c)

A 0.90 g (0.0019 mol) portion of 26c was dissolved in 10 mL of methanol, and 0.191 g (0.0059 mol) of hydrazine was added dropwise with stirring. The reaction was allowed to reflux under nitrogen for 12 h, and then the reaction mixture was concentrated in vacuo to yield a white semisolid. This solid was dissolved in 50 mL of 4.0 N ammonium hydroxide and extracted with three 50 mL portions of chloroform. The combined organic layers were dried over magnesium sulfate, filtered and concentrated in vacuo, to yield 27c (0.560 g, 88.3%) as a white amorphous solid. ¹H NMR (400 MHz CDCl₃) δ 1.37 (broad m, 2H), 1.45 (s, 9H), 1.47 (m, 2H), 2.6 (t, J = 7.2 Hz, 2H), 2.92 (s, 6H), 3.09 (broad m, 2H), 4.3 (s, 2H), 6.6 (d, 2H), 7.1 (m, 2H). ¹³C NMR (400 MHz CDCl₃) δ 26.19, 28.66, 37.85, 46.02, 53.65, 60.61, 79.82 123.41, 112.85, 127.36, 127.95, 168.59. IR (cm⁻¹) 3365.8, 2974.8, 2929.7, 1689.8, 1570.5, 1467.0, 1310.1, 1168.6.

1-N-[4-(Methyl)benzyl]-1-N-[(tert-butyloxy)carbonyl]-4-aminobutylamine (27b)

Compound 27b was synthesized from 26b according to the procedure used to synthesize 27c in 82.8% yield. ¹H NMR (400 MHz CDCl₃) δ 1.38 (m, 2H), 1.44–1.48 (m, 11H), 2.32 (s, 3H), 2.66 (t, J = 6.8 Hz, 2H), 3.11–3.19 (broad m, 2H), 4.38 (s, 2H), 7.11 (s, 4H). ¹³C NMR (400 MHz CDCl₃) δ 25.39, 28.67, 31.22, 42.13, 46.36.49.68, 50.13, 79.69, 115.28, 127.33, 127,92, 129.33, 135.67, 138.90. IR (cm⁻¹) 3360.6, 2975.8, 2929.3, 1690.8, 1514.9, 1410.6, 1365.3, 1245.0.

1-N-[2-(Phenyl)benzyl]-1-N-[(tert-butyloxy)carbonyl]-4-aminobutylamine (27a)

Compound 27a was synthesized from 26a according to the procedure used to synthesize 27c in 78.6% yield. ¹H NMR (400 MHz CDCl₃) δ 1.24–1.31(m, 4H), 1.461(s, 9H), 2.56(m, 2H), 2.95(broad s, 1H), 3.08(bs, 1H), 4.35–(s, 1H), 4.45(s, 1H), 7.31–7.41(m, 9H) ¹³C NMR (400 MHz CDCl₃) δ 25.32, 25.38, 28.56, 28.63, 31.08, 42.06, 46.19, 46.63, 48.05, 79.72, 126.97, 127.36, 127.85, 128.45, 129.36, 130.12, 141.06 IR (cm⁻¹) 3372.9, 2929.4, 1683.0, 1569.4, 1514.2, 1473.2, 1198.7.

5-{4-[N-(tert-Butyloxy)carbonyl]-4-[N,N-(dimethylamino)-benzyl]amino}butylcarbamoylpentanoic Acid Methyl Ester (29d)

A 0.500 g (0.0051 mol) portion of 27c was dissolved in 15 mL of dichloromethane, and the reaction mixture was cooled to 0 °C. Two to three drops of triethylamine was then added to the solution, and the reaction was allowed to stir for 15 min. The acid chloride 28a (0.333 g, 0.0019 mol) was slowly added to the reaction mixture, which was allowed to stir at 0 °C for 15 min, then heated to room temperature and allowed to stir for an additional 8 h. The solvent was removed in vacuo, and the residue was dissolved in water and extracted with three 50 mL portions of chloroform. The organic layers were combined and washed with 50 mL of saturated sodium bicarbonate and 50 mL of saturated sodium chloride and then dried over magnesium sulfate. Filtration and removal of the solvent in vacuo then afforded crude 29d. Purification on silica gel (hexane:EtOAc 1:3) then gave pure 29d (0.580 g, 80.2%) as a clear yellow oil. ¹H NMR (CDCl₃) δ 1.2 (m, 4H), 1.4 (s, 9H), 1.5 (m, 4H), 2.11 (t, J = 7.2 Hz, 2H), 2.28 (t, J = 7.6 Hz, 2H), 2.93 (s, 6H), 3.22 (m, 4H), 3.6 (s, 3H), 4.3 (s, 2H), 6.6 (d, J = 8.4 Hz, 2H), 7.1 (m, 2H).¹³C NMR (400 MHz CDCl₃) δ 24.64, 25.35, 26.32, 28.71, 33.89, 34.01, 36.41, 39.30, 40.92, 45.57, 50.06, 51.77, 79.72, 112.84, 128.66, 150.06, 156.17, 172.93, 174.21 IR (cm⁻¹) 3416.2, 2942.7, 1736.2, 1651.9, 1554.6, 1522.2, 1256.8.

7-{4-[(tert-Butyloxy)carbonyl]-[4-(methyl)benzyl]-amino}butylcarbamoylheptanoic Acid Methyl Ester (29e)

Compound 29e was synthesized from 27b and 28b according to the procedure used to synthesize 29d in 64.8% yield. ¹H NMR (400 MHz CDCl₃) δ 1.33 (m, 4H), 1.46 (broad s, 13H), 1.62 (m, 4H), 2.14 (m, 2H), 2.29 (m, 2H), 2.39 (s, 3H), 3.22 (m, 4H), 3.66 (s, 3H), 4.37 (s, 2H), 7.12 (s, 4H). ¹³C NMR (400 MHz CDCl₃) TM 24.94, 25.55, 25.77, 26.46, 28.66, 28.99, 29.08, 34.17, 36.85, 39.23, 46.04, 49.85, 50.4, 51.66, 80.01, 127.41,−127.94, 129.36, 135.55, 137.00, 156.13, 173.30, 174.41. IR (cm⁻¹) 3607.4, 3328.7, 2936.2, 2858.4, 1738.6, 1691.8, 1652.0, 1548.6, 1509.1.

7-{4-[(tert-Butyloxy)carbonyl]-4-[N,N-(dimethylamino)-benzyl]amino}butylcarbamoylheptanoic Acid Methyl Ester (29c)

Compound 29c was synthesized from 27c and 28b according to the procedure used to synthesize 29d in 76.5% yield. ¹H NMR (CDCl₃) δ 1.40–1.59 (m, 13H), 1.60 (m, 8H), 2.11 (m, 2H), 2.3 (m, 2H), 2.95 (s, 6H), 3.20 (m, 4H), 3.66 (s, 3H), 4.23 (s, 2H), 6.69 (d,J = 8 Hz, 2H), 7.11(broad s, 2H). ¹³C NMR (400 MHz CDCl₃) δ 24.64, 25.35, 26.32, 28.25, 28.64, 28.71, 29.07, 33.89, 35.98, 36.88, 40.01, 41.13, 46.57, 50.07, 51.92, 80.02, 128.66, 129.50, 150.06, 156.17, 172.93, 174.21. IR (cm⁻¹) 3437.5, 2956.6, 1739.3, 1651.0, 1550.6, 1522.2, 1256.8.

5-{4-[(tert-Butyloxy)carbonyl]-[4-(methyl)benzyl]-amino}butylcarbamoylpentanoic Acid Methyl Ester (29b)

Compound 29b was synthesized from 27b and 28a according to the procedure used to synthesize 29d in 70.7% yield. ¹H NMR (400 MHz CDCl₃) δ 1.44 (broad s, 13H), 1.60 (m, 4H), 2.14 (m, 2H), 2.29 (m, 2H), 2.4 (s, 3H), 3.22 (m, 4H), 3.67 (s, 3H), 4.33 (s, 2H), 7.12 (s, 4H). ¹³C NMR (400 MHz CDCl₃) δ 25.77, 26.46, 28.99, 29.08, 34.17, 36.85, 39.23, 46.04, 49.85, 50.4, 51.66, 80.01, 127.41,127.94, 129.36, 135.55, 137.00, 156.13, 173.30, 174.41. IR (cm⁻¹) 3338.4, 3318.9, 2936.2, 2858.4, 1736.2, 1690.8, 1651.9, 1548.1, 1509.1.

5-{4-[(tert-Butyloxy)carbonyl]-[2-(phenyl)benzyl]-amino}butylcarbamoyl}pentanoic Acid Methyl Ester (29a)

Compound 29a was synthesized from 27a and 28a according to the procedure used to synthesize 29d in 62.4% yield. ¹H NMR (400 MHz CDCl₃) δ 1.23–1.48 (m,13H), 1.62–1.71 (m, 4H), 2.15 (m, 2H), 2.31–2.38 (m, 2H), 2.96 (s, 1H), 3.08 (m, 4H), 3.64 (s, 3H), 4.35 (s, 1H), 4.43 (s, 1H), 6.26 (s, 1H), 7.20–7.42 (m, 9H). ¹³C NMR (400 MHz CDCl₃) δ 24.86, 28.33, 36.502, 39.35, 46.12, 47.14, 48.15, 51.73, 51.78, 60.60, 79.97, 126.76, 126.92, 127.41, 127.59, 127.85, 128.49, 129.31, 130.15, 172.93, 173.48, 174.13, 175.72. IR (cm⁻¹) 3304.9, 2921.0, 1722.3, 1671.4, 1651.9., 1551.6, 1158.9.

5-{4-[N-(tert-Butyloxy)carbonyl]-4-[N,N-(dimethylamino)-benzyl]amino}butylcarbamoylpentanoic Acid (30d)

A 0.500 g (0.0011 mol) portion of 29d was dissolved in 6 mL of tetrahydrofuran:water (4:2) and cooled to 0 °C, and 6 mL of 1.0 N LiOH was added to the mixture by dropwise addition. The solution was warmed to room temperature and allowed to stir for 16 h, during which time the reaction was monitored by TLC. The mixture was again cooled to 0 °C, neutralized by the dropwise addition of 2.0 N HCl, and extracted with three 50 mL portions of ethyl acetate. The ethyl acetate layers were combined, washed with brine, and dried over anhydrous magnesium sulfate. Removal of the solvent in vacuo yielded compound 30d as a cloudy yellow oil (0.375 g, 75.8%) of sufficient purity to be used in the next reaction without further purification. ¹H NMR (400 MHz CDCl₃) δ 1.2 (m, 4H), 1.4 (s, 9H), 1.5 (m, 4H), 2.11 (t, J = 7.2 Hz, 2H), 2.28 (t, J = 7.2 Hz, 2H), 2.93 (s, 6H), 3.22 (m, 4H), 4.3 (s, 2H), 6.6 (d, J = 8.8 Hz, 2H), 7.1 (m, 2H). ¹³C NMR (400 MHz CDCl₃) δ 21.29, 24.57, 25.20, 26.35, 28.72, 29.64, 29.92, 36.19, 39.26, 40.99, 45.76, 50.09, 60.65, 80.01, 113.01, 128.68, 150.09, 171.45, 172.28. IR (cm⁻¹) 3325.4, 2929.7, 2858.4, 1716.8, 1658.4, 1613. 0, 1554.6, 1486.8, 1006.0.

7-{4-[N-(tert-Butyloxy)carbonyl]-4-[4-(methyl)benzyl]-amino}butylcarbamoylheptanoic Acid (30e)

Compound 30e was synthesized from 29e according to the procedure used to synthesize 30d in 72.7% yield. ¹H NMR (400 MHz CDCl₃) δ 1.18 (m, 6H), 1.39 (bs, 12H), 1.53 (m, 4H), 2.10 (m, 2H), 2.2 (m, 2H), 2.26 (s, 3H), 3.14 (m, 4H), 4.30 (s, 2H), 7.05 (s, 4H) ¹³C NMR (400 MHz CDCl₃) δ, 21.93, 24.93, 25.70, 26.44, 28.62, 28.87, 29.01, 31.07, 36.6, 39.26, 46.05, 49.73, 50.35, 60.58, 64.53, 80.09, 127.34, 127.85, 129.33, 135.45, 136.93, 156.1, 171.38, 173.91. IR (cm⁻¹) 3326.9, 2932.8, 2863.1, 1730.5, 1691.0, 1644.1, 1661.8, 1555.3, 1464.62, 1366.05, 1244.7, 1168.19, 1036.0, 729.15 (cm⁻¹)

7-{4-[N-(tert-Butyloxy)carbonyl]-4-[N,N-(dimethylamino)-benzyl]amino}butylcarbamoylheptanoic Acid (30c)

Compound 30c was synthesized from 29c according to the procedure used to synthesize 30d in 72.3% yield. ¹HNMR (400 MHz CDCl₃) δ 1.35–1.44 (m, 17H), 1.63 (m, 4H), 2.2 (m, 4H), 2.32 (m, 2H), 2.93 (s, 6H), 3.16 (m, 4H), 4.29 (s, 2H), 6.69 (d, J = 8.8 Hz, 2H), 7.11 (m, 2H). ¹³C NMR (400 MHz CDCl₃) δ 14.34, 21.19, 21.93, 24.93, 25.46, 25.76, 26.43, 28.60, 28.88, 29.01, 31.07, 36.6, 39.26, 46.05, 49.73, 50.35, 60.58, 64.53, 80.09, 127.34, 127.85, 129.33, 135.45, 136.93, 156.1, 171.38, 173.91. IR (cm⁻¹) 3348.2, 2929.7, 1718.3, 2658.4, 1616.4, 1555.6, 1478.8, 1398.1, 1110.7.

5-{4-[(tert-Butyloxy)carbonyl]-[4-(methyl)benzyl]-amino}butylcarbamoylpentanoic Acid Methyl Ester (30b)

Compound 30b was synthesized from 29b according to the procedure used to synthesize 30d in 70.9% yield. ¹H NMR (400 MHz CDCl₃) δ 1.2(m, 4H), 1.40 (broad s, 9H), 1.53 (m, 4H), 2.10 (m, 2H), 2.2 (m, 2H), 2.30 (s, 3H), 3.20 (m, 4H), 4.32 (s, 2H), 7.05 (s, 4H). ¹³C NMR (400 MHz CDCl₃) δ 25.70, 26.44, 28.62, 28.87, 29.01, 31.07, 36.6, 39.26, 46.05, 49.73, 50.35, 60.58, 64.53, 80.09, 127.34, 127.85, 129.33, 135.45, 136.93, 156.1, 171.38, 173.91. IR (cm⁻¹) 3326.4, 2934.4, 2863.1, 1729.7, 1691.7, 1645.4, 1664.8, 1555.3, 1464.6, 1421.1, 1168.2, 1136.0, 732.4.

5-{4-[(tert-Butyloxy)carbonyl]-[2-(phenyl)benzyl]-amino}butylcarbamoyl}pentanoic Acid Methyl Ester (30a)

Compound 30a was synthesized from 29a according to the procedure used to synthesize 30d in 86.1% yield. ¹H NMR (400 MHz CDCl₃) δ 1.30–1.43 (m,13H),1.54 (m,4H), 2.11–2.27 (m,4H), 2.93–3.05 (m,4H), 4.36(d, J = 32 Hz, 2H), 7.20–7.38 (m,9H). ¹³C NMR (400 MHz CDCl₃) δ 21.29, 25.58, 26.54, 28.56, 29.68, 29.92, 30.54, 30.85, 36.26, 39.32, 46.38, 47.24, 48.27, 60.64, 64.60, 80.10, 125.75, 126.77, 127.05, 127.44, 127.85, 128.53, 129.3, 130.18, 135.51, 135.83, 140.97, 141.56, 156.03, 173.62. IR (cm⁻¹) 3304.9, 2921.0, 1730.0, 1671.4, 1651.9, 1551.6, 1158.9.

5-{4-[N-(tert-Butyloxy)carbonyl]-4-[N,N-(dimethylamino)-benzyl]amino}butylcarbamoyl-pentanohydroxamic Acid (31d)

Ethyl chlorformate (0.086 g, 0.0008 mol) and triethylamine (0.08 g, 0.0008 mmol) were added to the solution of 30d (0.300 g, 0.0007 mol) in 5 mL of THF, and the mixture was cooled to 0 °C. The reaction was allowed to stir for 20 min, after which time it was filtered and added to 20 mL of freshly prepared 1.76 M hydroxylamine in methanol. The reaction was stirred at room temperature for 30 min, filtered and concentrated in vacuo to yield the crude 31d. Purification on silica gel (ethyl acetate:methanol 4:2) then afforded pure 31d (0.252 g, 77.5%) as a dark yellow oil. ¹H NMR (400 MHz CDCl₃) δ 1.2 (m, 4H), 1.4 (s, 9H), 1.5 (m, 4H), 2.15 (broad s, 4H), 2.93 (s, 6H), 3.22 (m, 4H), 3.6 (s, 3H), 4.3 (s, 2H), 6.6 (d, J = 8.0 Hz, 2H), 7.1 (m, 2H), 7.8 (broad s, NHOH). ¹³C NMR (400 MHz CDCl₃) δ 21.2, 23.11, 25.71, 28.72, 31.51, 39.45, 40.91, 41.08, 45.79, 112.86, 1125.73, 126.25, 128.37, 128.62, 132.28, 150.12, 156.13. IR (cm⁻¹) 3396.8, 2929.7, 1638.9, 1610.2,1554.6, 1450.8, 1405.4.

7-{4-[N-(tert-Butyloxy)carbonyl]-4-[4-(methyl)benzyl]-amino}butylcarbamoylheptanohydroxamic Acid (31e)

Compound 31e was synthesized from 30e according to the procedure used to synthesize 31d in 78.0% yield. ¹H NMR (400 MHz CDCl₃) δ 1.24–1.31 (m, 4H), 1.43–1.6 (m, 13H), 2.16 (m, 2H), 2.32 (s, 3H), 3.18 (m, 4H), 4.35 (s, 2H), 7.11 (s, 4H). ¹³C NMR (400 MHz CDCl₃) δ 20.07, 23.29, 25.29, 25.46, 25.57, 26.38, 28.53, 28.61, 32.45, 35.71, 38.24, 46.88, 50.92, 80.01, 117,7, 128.33, 129.64, 129.74, 139.67, 156.22, 174.0.38. IR (cm⁻¹) 3221.6, 2929.7, 2858.4, 1671.4, 1638.9, 1554.6, 1450.8, 1190.1, 1150.8, 1110.9.

7-{4-[N-(tert-Butyloxy)carbonyl]-4-[N,N-(dimethylamino)-benzyl]amino}butylcarbamoyl-heptanohydroxamic Acid (31c)

Compound 31c was synthesized from 30c according to the procedure used to synthesize 31d in 75.7% yield. ¹H NMR (400 MHz CDCl₃) δ 1.24 (m, 4H), 1.44(m, 15H), 1.52 (m, 2H), 2.1 (m, 4H), 2.90 (s, 6H), 3.14 (m, 4H), 4.26 (s, 2H), 6.65 (d, J = 7.6 Hz, 2H), 7.08 (broad s, 1H). ¹³C NMR (400 MHz CDCl₃) δ 21.34, 22.43, 23.11, 25.71, 28.72, 31.51, 39.45, 40.91, 41.08, 45.79, 112.86, 1125.73, 126.25, 128.37, 128.62, 132.28, 150.12, 156.13. IR (cm⁻¹) 3385.7, 2932.4, 1632.4, 1622.0.0, 1554.6, 1460.7, 1425.1, 1304.2.

5-{4-[N-(tert-Butyloxy)carbonyl]-4-[4-(methyl)benzyl]-amino}butylcarbamoylpentanohydroxamic Acid (31b)

Compound 31b was synthesized from 30b according to the procedure used to synthesize 31d in 80.9% yield. ¹H NMR (400 MHz CDCl₃) δ 1.24–1.31 (m, 4H), 1.43 (m, 9H), 2.18 (m, 4H), 2.32 (s, 3H), 3.18 (m, 4H), 4.32 (s, 2H), 7.11 (s, 4H). ¹³C NMR (400 MHz CDCl₃) δ 25.31, 25.06, 26.38, 28.53, 28.61, 32.45, 35.71, 38.24, 46.88, 50.92, 80.24, 127.37, 127.9, 129.64, 129.74, 137.67, 156.33, 174.21. IR (cm⁻¹) 3267.0, 2929.7, 2858.4, 1651.9, 1548.1, 1463.8, 1418.4.

5-{4-[(tert-Butyloxy)carbonyl]-4-[2-(phenyl)benzyl]-amino}butylcarbamoyl}pentanohydroxamic Acid (31a)

Compound 31a was synthesized from 30a according to the procedure used to synthesize 31d in 80.6% yield. ¹H NMR (400 MHz CDCl₃) δ 1.25–1.42(m,15H), 1.56 (m,2H),1.99–2.26 (m,−4H),2.94–3.05(m,4H), 4.36(d, J = 31.2 Hz, 2H),7.21–7.40(m,−9H) ¹³C NMR (400 MHz CDCl₃) δ 25.04, 25.49, 26.47, 28.57, 29.92, 32.24, 35.91, 39.98, 46.5, 48.32, 80.24, 126.8, 125.08, 127.47, 127.88, 128.55, 129.3, 130.2, 135.74, 140.95, 141.58, 156.18, 174.09. IR (cm⁻¹) 3260.5, 2975.1, 2929.7, 1664.9, 1651.9, 1554.6.

5-{4-[4-N,N-(Dimethyl)aminobenzyl]amino}butyl-carbamoylpentanohydroxamic Acid (17)

Compound 31d (0.140 g, 0.0003 mol) was dissolved in 5 mL of 20% trifluoroacetic acid in dichloromethane, and the reaction was allowed to stir at room temperature for 8 h. The solvent was then removed in vacuo, and the residue was taken up in 25 mL of chloroform and dried over anhydrous magnesium sulfate. Filtration and removal of the solvent afforded crude 17 (0.110 g, 76.7%) as a buff colored solid. An analytical sample of 17 was prepared by recrystallization from aqueous ethanol. ¹H NMR (400 MHz CD₃OD) δ 1.2 (m, 4H), 1.5 (m, 4H), 2.15 (t, J = 7.2 Hz, 2H), 2.19 (t, J = 7.2 Hz, 2H), 2.93 (s, 6H), 3.22 (m, 4H), 4.03 (s, 2H), 6.7(d, J = 8.0 Hz, 2H), 7.3 (m, 2H). ¹³C NMR (400 MHz CD₃OD) δ 23.28, 24.35, 26.31, 131.63, 161.58. IR (cm⁻¹) 2988.1, 1671.4, 1554.6, 1444.3, 1203.8, 1165.9. Anal. for C₂₁H₃₃F₃N₄O₅: C, H, N.

7-{4-[4-(Methyl)benzyl]aminobutyl}-carbamoylheptanohydroxamic Acid (18)

Compound 18 was synthesized from 31e according to the procedure used to synthesize 17 in 71.6% yield. An analytical sample of 18 was prepared by recrystallization from aqueous ethanol. ¹H NMR (400 MHz CD₃OD) δ 1.23–1.43 (m, 4H), 1.59–1.6 (m, 8H), 2.07–2.17 (m, 4H), 2.35 (s, 3H), 2.86 (t, J = 7.2 Hz, 2H), 3.03 (m, 2H), 3.19 (m, 2H), 4.14 (s, 2H), 7.26 (d, J = 6.4, 2H), 7.37 (d, J = 6.8 Hz, 2H), 8.06 (broad s, NHCO), 9.93(broad s, NHOH). ¹³C NMR (400 MHz CD₃OD) δ 23.3, 25.4, 25.7, 26.44, 28.54, 28.62, 32.43, 35.68, 38.27, 50.82, 117.70, 128.33, 129.64, 129.8, 139.7, 160.41, 175.21 IR (cm⁻¹) 3226.6, 2929.7, 2853.4, 1671.4, 1640.3, 1551.0, 1444.7, 1209.8, 1163.8. Anal. for C₂₂H₃₄F₃N₃O₅: C, H, N.

7-{4-[4-N,N-(Dimethylamino)benzyl]aminobutyl}-carbamoylheptanohydroxamic Acid (13)

Compound 13 was synthesized from 31c according to the procedure used to synthesize 17 in 72.1% yield. An analytical sample of 13 was prepared by recrystallization from aqueous ethanol. ¹H NMR (400 MHz CD₃OD) δ 1.62 (m, 8H), 1.74 (m, 2H), 2.12 (t, J = 6.8 Hz, 2H), 2.21 (t, J = 6.8 Hz, 2H), 3.06 (m, 2H), 3.15 (s, 6H), 3.21 (m, 2H), 4.20 (s, 2H), 7.36 (broad s, 2H), 7.58 (broad s, 2H). ¹³C NMR (400 MHz CD₃OD) δ 26.86, 26.9,28.12, 29.01, 29.2, 29.47, 30.13, 30.35, 36.19, 43.09, 46.58, 44.4, 57.0, 112.8, 112.9, 129.32, 129.4, 168.56, 174.23 IR (cm⁻¹) 2982.2, 1671.4, 1556.1, 1445.3, 1203.8, 1168.4. Anal. for C₂₃H₃₇F₃N₄O₅: C, H, N.

5-{4-[4-(Methyl)benzyl]aminobutyl}carbamoylpentanohydroxamic Acid (8)

Compound 8 was synthesized from 31b according to the procedure used to synthesize 17 in 72.9% yield. An analytical sample of 8 was prepared by recrystallization from aqueous ethanol. ¹H NMR (400 MHz CD₃OD) δ 1.23–1.43 (m, 4H), 1.6 (m, 4H), 2.0–2.1 (m, 4H), 2.35 (s, 3H), 2.86 (t, J = 7.2 Hz, 2H), 3.02 (m, 2H), 3.20 (m, 2H), 4.23 (s, 2H), 7.24 (d, J = 6.4, 2H), 7.35 (d, J = 6.8 Hz, 2H). ¹³C NMR (400 MHz CD₃OD) δ 26.04, 28.63, 28.54, 34.86, 36.12, 38,99, 51.32, 118.48, 128.33, 129.64, 129.8, 139.7, 160.14, 175.21. IR (cm⁻¹) 3221.6, 2929.7, 2858.4, 1781.6, 1671.4, 1638,9, 1554.6, 1450.8, 1190.1, 1150.8, 1110.9. Anal. for C₂₀H₃₀F₃N₃O₅: C, H, N.

5-{4-[2-(Phenyl)benzyl]aminobutyl}carbamoylpentanohydroxamic Acid (6)

Compound 6 was synthesized from 31a according to the procedure used to synthesize 17 in 87% yield. An analytical sample of 6 was prepared by recrystallization from aqueous ethanol. ¹H NMR (400 MHz CD₃OD) δ 1.53(m,−4H),1.60(m,4H),2.10(m,2H),2.18(m,2H),2.82(t, J = 8 Hz, 2H),3.11 (t, J = 6.4 Hz, 2H),4.22 (s,2H),7.35(d, J = 7.2 Hz, 3H), 7.36–7.50(m,5H), 7.66(d, J = 2.8 Hz, 1H) ¹³C NMR (400 MHz CD₃OD) δ 22.92, 24.9, 25.25, 25.47, 26.2, 32.11, 35.45, 38.10, 47.02, 127.82, 128.31, 1128.70, 128.96, 129.29, 129.47, 130.73, 139.95, 143.27, 155.38, 171.12, 174.79. IR (cm⁻¹) 3723.51, 2923.24, 2851.89, 1671.35, 1561.08, 1431.35, 1176.6, 1112.7. Anal. for C₂₅H₃₂F₃N₃O₅: C, H, N.

1-Phthalimido-4-{N-[2,4,6-(trimethyl)benzenesulfonyl]}-8-{N-[4-(tert-butyl)benzyl]-N-[2,4,6-(trimethyl)-benzenesulfonyl]}amino-4-azaoctane (33a)

Sodium hydride 60% oil dispersion (equivalent to 0.027 g, 0.0011 mol of NaH was dissolved in 1 mL of dimethylformamide under nitrogen, and the reaction was cooled to 0 °C. Compound 32 (0.500 g, 0.0010 mol) was dissolved in 4 mL of dimethylformamide and added dropwise to the reaction mixture, which was allowed to stir for 30 min. A 0.250 g portion (0.0011 mol) of 4-tert-butylbenzyl bromide was dissolved in 2 mL of dimethylformamide and added to the reaction slowly via syringe. The reaction was stirred for 12 h, the solvent was removed in vacuo and the residue was dissolved in water and extracted with three 50 mL portions of ethyl acetate. The combined organic layers were dried over anhydrous magnesium sulfate and filtered, and the solvent was removed to yield crude 33a. Purification of the crude material on silica gel (hexane:ethyl acetate 4: 3) then afforded pure 33a as a fluffy white solid (0.619 g, 71.6%). ¹H NMR (400 mHz CDCl₃) δ 1.25 (s, 9H) 1.4 (m, 2H), 1.61 (m,2H), 1.72 (m, 2H), 2.24 (s, 6H), 2.62 (s, 6H), 2.62 (s, 6H), 3.04–3.06 (t, J = 6.4 Hz, 4H), 3.18 (t, J = 7.2 Hz, 2H), 4.17 (s,2H), 6.95 (s, 2H), 7.23 (d, J = 6.8 Hz, 2H), 7.73 (m, 2H), 7.83–7.85 (m, 2H). ¹³C NMR (400 MHz CDCl₃) δ 21.19, 23.06, 24.32, 24.49, 24.66, 25.29, 25.39, 25.95, 27.11, 28.70, 31.52, 33.78, 33.87, 36.28, 36.40, 36.47, 43.52, 43.64, 51.76, 51.89, 132.15, 132.30, 132.34, 133.18, 140.15, 140.27, 142.92, 173.24, 174.02, 174.17, 178.84.

1-Phthalimido-4-{N-[2,4,6-(trimethyl)benzenesulfonyl]}-8-{N-[3,3-(diphenyl)propyl]-N-[2,4,6-(trimethyl)-benzenesulfonyl]}amino-4-azaoctane (33b)

Compound 33b was synthesized from 32 and 3,3-diphenylpropyl chloride using the procedure described for the synthesis of 33a in 60.3% yield. ¹H NMR (400 MHz CDCl₃) δ 1.40 (m, 4H), 1.69 (q, J = 7.2 Hz, 2H), 2.08 (q, J = 8 Hz, 2H), 2.3 (s, 3H), 2.44 (s, 3H), 2.51 (d, J = 11.2 Hz, 12H), 3.03 (t, J = 8 Hz, 2H), 3.09 (t, J = 8 Hz, 1H), 3.16 (m, 4H), 3.46 (t, J = 6.8 Hz, 2H), 3.73 (t, J = 8 Hz, 1H), 6.76 (s, 2H), 6.90 (s, 2H), 7.03 (d, J = 7.6 Hz, 2H), 7.12–7.27 (m, 7H), 7.68 (m, 2H), 7.8 (m, 2H). ¹³C NMR (400 MHz CDCl₃) δ 14.43, 21.18, 21.20, 21.29, 22.96, 22.99, 24.85, 24.90, 26.74, 32.98, 35.43, 44.37, 44.42, 45.40, 45.44, 48.96, 60.62, 123.40, 126.59, 127.76, 128.78, 132.13, 132.18, 132.19, 133.09, 133.4, 134.23, 140.18, 140.35, 142.49, 142.66, 143.99, 168.25. IR (cm⁻¹) 3059.5, 3020.5, 2936.2, 1768.7, 1716.8, 1600.0, 1561.1, 1450.8, 1139.5.

1-Amino-4-{N-[2,4,6-(trimethyl)benzenesulfonyl]}-8-{N-[4-(tert-butyl)benzyl]-N-[2,4,6-(trimethyl)benzenesulfonyl]}amino-4-azaoctane (34a)

Compound 34a was prepared from 33a using the procedure described for the synthesis of 27c in 70.8% yield. ¹H NMR (CDCl₃) δ 1.28(s,9H) 1.4 (m, 2H), 1.61 (m,2H), 1.56 (m, 2H), 2.24 (s, 6H), 2.51 (s, 6H), 2.57 (s,−6H), 2.62 (q, J = 6.8 Hz, 2H), 3.08 (t, J = 7.2 2H), 3.24 (t, J = 7.6 Hz, 2H), 3.49 (t, J = 7.2 Hz, 2H), 4.2 (s,2H), 6.81 (s, 2H), 6.95 (m, 6H), 7.23 (d, J = 6.8 Hz, 2H). ¹³C NMR (400 MHz CDCl₃) δ 21.11, 21.48, 21.79, 22.5, 22.8, 25.42, 26.87, 28.43, 28.48, 31.74, 32.59, 40.08, 46.77, 46.81, 125.12, 128.2, 136.44, 145.1, 145.64.

1-Amino-4-{N-[2,4,6-(trimethyl)benzenesulfonyl]}-8-{N-[3,3-(diphenyl)propyl]-N-[2,4,6-(trimethyl)benzenesulfonyl]}amino-4-azaoctane (34b)

Compound 34b was prepared from 33b using the procedure described for the synthesis of 27c in 78.5% yield. ¹H NMR (400 MHz CDCl₃) δ 1.37 (m, 4H), 1.58 (q, J = 7.2 Hz, 2H), 2.04 (m, 2H), 2.28 (s, 3H), 2.31 (s, 3H), 2.46 (s, 6H), 2.57 (s, 6H), 2.97 (t, J = 8 Hz, 2H), 3.10 (t, J = 4.8 Hz, 2H), 3.17 (m, 4H), 3.48 (t, J = 7.2 Hz, 1H), 3.7 (q, J = 5.6 Hz, 2H), 6.9 (d, J = 3.2 Hz, 4H), 7.01 (d, J = 6.8 Hz, 4H), 7.12–7.23 (m, 5H). ¹³C NMR (400 MHz CDCl₃) δ 14.40, 14.99, 15.84, 21.14, 21.19, 21.26, 22.91, 23.05, 24.66, 25.33, 25.44, 29.90, 31.02, 39.31, 41.28, 42.45, 43.20, 44.63, 45.14, 45.46, 48.90, 52.04, 55.64, 60.06, 64.29, 126.60, 127.67, 128.76, 129.46, 132.18, 133.35, 133.40, 140.22, 140.29, 142.51, 142.60, 143.89, 149.41 IR (cm⁻¹) 3364.3, 3027.0, 2936.2, 2871.4, 1600.0, 1561.1, 1314.6, 1139.5.

15-{N-[2,4,6-(Trimethyl)benzenesulfonyl-N-[(4-tert-butyl)benzyl]}amino-11-[N-2,4,6-(trimethyl)benzene-sulfonyl]-6-oxo-7,11-diazapentadecanoic Acid Methyl Ester (35a)

Compound 35a was synthesized from 34a and 27a using the method described for the synthesis of 29c in 72.3% yield. ¹H NMR (CDCl₃) δ 1.28 (s, 9H) 1.34 (m, 4H), 1.61 (m, 4H), 1.72 (m, 2H), 2.11 (t, J = 7.2 Hz, 2H), 2.24 (m, 8H), 2.51 (s, 6H), 2.62 (s, 8H), 3.08 (m, 2H), 3.24 (m, 2H), 3.6 (t, 3H), 4.13 (s,2H), 6.90 (broad s, 2H), 7.22 (broad s, 2H), 7.28 (d, 2H). ¹³C NMR (400 MHz CDCl₃) δ 14.35, 14.41, 21.19, 21.27, 22.87, 23.11, 23.12, 24.17, 24.46, 24.68, 25.29, 27.12, 31.50, 31.8, 33.91, 34.72, 36.34, 36.38, 43.29, 44.57, 45.12, 48.93, 51.75, 60.61, 125.74, 128.38, 132.28, 132.30, 132.56, 133.36, 133.39, 140.25, 140.29, 142.80, 151.11, 172.95, 174.11 IR (cm⁻¹) 3387.9, 2952.2, 1738.3, 1655.3, 1603.5, 1541.2, 1458.2.

17-{N-[2,4,6-(Trimethyl)benzenesulfonyl-N-[(4-tert-butyl)benzyl]}amino-13-[N-2,4,6-(trimethyl)benzene-sulfonyl]-8-oxo-7,11-diazaheptadecanoic Acid Methyl Ester (35b)

Compound 35b was synthesized from 34a and 27b using the method described for the synthesis of 29c in 78.3% yield. ¹H NMR (400 MHz CDCl₃) δ 1.28 (s, 9H), 1.32–1.36 (m, 8H), 1.58–1.7 (m, 8H), 2.09 (t,J = 2.8 Hz, 2H), 2.30 (m, 8H), 2.58 (d, J = 9.2 Hz, 12H), 3.03 (m, 4H), 3.20 (q, J = 6.8 Hz, 6H), 3.6 (s, 3H), 4.12 (s, 2H), 5.979 (t, J = 5.6 Hz, 1H), 6.90 (d, J = 8.4 Hz, 2H), 6.96 (s, 4H), 7.27 (d, J = 1.2 Hz, 2H). ¹³C NMR (400 MHz CDCl₃) δ 21.19, 23.12, 24.17, 24.46, 24.74, 24.93, 24.97, 25.74, 27.09, 28.89, 28.95, 29.02, 29.10, 31.51, 34.06, 34.2, 34.73, 36.36, 36.76, 43.29, 44.58, 45.13, 48.95, 21.70, 51.72, 125.74, 128.37, 132.28, 132.55, 133.36, 133.39, 140.25, 140.31, 142.81, 151.13, 173.66, 174.45, 178.43. IR (cm⁻¹) 2929.7, 2864.9, 1736.2, 1645.4, 1600.0, 1554.6, 1314.6, 1139.5.

17-{N-[2,4,6-(Trimethyl)benzenesulfonyl-N-[3,3-(diphenyl)propyl]}amino-13-[N-2,4,6-(trimethyl)-benzenesulfonyl]-8-oxo-7,11-diazaheptadecanoic Acid Methyl Ester (35c)

Compound 35c was synthesized from 34b and 27b using the method described for the synthesis of 29c in 75.1% yield. ¹H NMR (400 MHz CDCl₃) δ 1.25–1.39 (m, 8H), 1.55–1.69 (m, 6H), 2.05 (q, J = 8 Hz, 2H), 2.07 (m, 2H), 2.28 (m, 2H), 2.32 (m, J = 8 Hz, 8H), 2.45 (s, 6H), 2.57 (s, 6H), 2.93 (t, J = 7.6 Hz, 2H), 3.10 (t, J = 7.6 Hz, 2H), 3.15–3.22 (m, 6H), 3.65 (s, 3H), 3.69 (t, J = 8 Hz, 1H), 5.84 (t, 1H), 6.91 (d, J = 9.6 Hz, 2H), 6.98 (d, J = 7.2 Hz, 4H), 7.14–7.20 (m, 5H). ¹³C NMR (400 MHz CDCl₃) δ 14.42, 21.17, 21.20, 21.29, 22.93, 23.10, 24.47, 24.72, 24.98, 25.73, 27.13, 29.04, 29.12, 32.75, 34.20, 36.35, 36.74, 43.32, 44.09, 45.14, 45.29, 48.87, 51.69, 60.29, 126.65, 127.65, 128.79, 132.24, 132.31, 133.36, 140.25, 142.61, 142.79, 143.82, 173.42, 174.42 IR (cm⁻¹) 2936.2, 2851.9, 1736.2, 1671.4, 1638.9, 1593.5, 1146.0.

15-{N-[2,4,6-(Trimethyl)benzenesulfonyl-N-[3,3-(diphenyl)propyl]}amino-11-[N-2,4,6-(trimethyl)-benzenesulfonyl]-6-oxo-7,11-diazapentadecanoic Acid Methyl Ester (35d)

Compound 35d was synthesized from 34b and 27a using the method described for the synthesis of 29c in 72.6% yield. ¹H NMR (400 MHz CDCl₃) δ 1.32–1.39 (m, 4H), 1.58–1.60 (m, 4H), 1.68 (q, J = 6.8 Hz, 4H), 1.99 (q, J = 8 Hz, 2H), 2.10 (t, J = 6.8 Hz, 2H), 2.28–2.35 (d, J = 9.2 Hz, 8H), 2.45 (s, 6H), 2.57 (s, 6H), 2.93 (t, J = 8 Hz, 2H), 3.09 (t, J = 9.6 Hz, 2H), 3.15–3.22 (m, 6H), 3.65 (m, 4H), 5.94 (s. 1H), 6.91 (d, J = 9.6 Hz, 4H), 6.93 (d, J = 6.8 Hz, 4H), 7.12–7.22 (m, 5H). ¹³C NMR (400 MHz CDCl₃) δ 14.41, 21.16, 21.19, 21.83, 22.92, 23.09, 24.46, 24.66, 24.71, 25.28, 27.14, 32.74, 33.91, 36.31, 36.42, 43.34, 44.08, 45.14, 45.28, 48.86, 51.75, 60.62, 126.64, 127.64, 128.79, 132.25, 132.31, 133.36, 140.23, 142.62, 142.81, 143.82, 172.96, 174.12. IR (cm⁻¹) 3370.8, 2929.7, 1736.2, 1671.4, 1651.9, 1600.0, 1541.6.

15-{N-[2,4,6-(Trimethyl)benzenesulfonyl-N-[(4-tert-butyl)benzyl]}amino-11-[N-2,4,6-(trimethyl)benzene-sulfonyl]-6-oxo-7,11-diazapentadecanoic Acid (36a)

Compound 36a was synthesized from 35a using the method described for the synthesis of 30d in 70.6% yield. ¹H NMR (CDCl₃) δ 1.28 (s,9H) 1.34 (m, 4H), 1.61 (m,4H), 1.72 (m, 2H), 2.11 (t, J = 8.0 Hz, 2H), 2.24 (m, 8H), 2.51 (s, 6H), 2.62 (s,−8H), 3.08 (m, 2H), 3.24 (m, 2H), 4.13 (s, 2H), 6.90 (broad s, 2H), 7.22 (broad s, 2H), 7.28 (d, J = 6.8 Hz, 2H). ¹³C NMR (400 MHz CDCl₃) δ 14.41, 21.27, 23.11, 14.71, 24.46, 24.11, 27.09, 28.97, 29.11, 30.89, 31.50, 34.35, 34.67, 36.54, 36.26, 43.22, 44.05, 45.61, 48.69, 60.46, 64.76, 125.17, 128.53, 132.72,132.73, 132.15, 133.73, 140.12, 140.43, 142.43, 142.88, 151.81, 171.34, 173.28 IR (cm⁻¹) 3377.3, 2929.7, 1723.2, 1645.4, 1600.0, 1548.1.

17-{N-[2,4,6-(Trimethyl)benzenesulfonyl-N-[(4-tert-butyl)benzyl]}amino-13-[N-2,4,6-(trimethyl)benzene-sulfonyl]-8-oxo-7,11-diazaheptadecanoic Acid (36b)

Compound 36b was synthesized from 35b using the method described for the synthesis of 30d in 80.0% yield. ¹H NMR (400 MHz CDCl₃) δ 1.27 (s,9H), 1.32 (m, 8H), 1.56–1.62 (m, 4H), 1.68 (t, J = 7.6 Hz, 2H), 2.30 (d, J = 5.6 Hz, 8H), 2.57 (d, J = 10 Hz, 2H), 3.01 (m, 4H), 3.02–3.23 (m, 4H), 4.12 (s, 2H), 6.05 (s,1H), 6.90 (d, J = 8 Hz, 2H), 6.95 (d, J = 3.2 Hz, 4H), 7.25 (d, J = 8.4 Hz, 2H).¹³C NMR (400 MHz CDCl₃) δ 13.92, 14.41, 19.34, 21.19, 21.27, 23.11, 23.12, 24.17, 24.46, 24.81, 25.68, 27.09, 28.91, 28.99, 29.67, 29.91, 30.83, 31.51, 34.37, 34.72, 36.42, 36.69, 43.32, 44.58, 45.13, 48.94, 60.64, 64.60, 125.74, 128.38, 132.28, 132.31, 132.56, 133.37, 140.25, 140.34, 142.81, 151.11, 171.45, 173.81 IR (cm⁻¹) 3370.9, 2929.7, 1723.2, 1645.4, 1600.0, 1549.2.

17-{N-[2,4,6-(Trimethyl)benzenesulfonyl-N-[3,3-(diphenyl)propyl]}amino-13-[N-2,4,6-(trimethyl)-benzenesulfonyl]-8-oxo-7,11-diazaheptadecanoic Acid (36c)

Compound 36c was synthesized from 35c using the method described for the synthesis of 30d in 72.8% yield. ¹H NMR (400 MHz CDCl₃) δ 1.25–1.43 (m, 8H), 1.58–1.67 (m, 6H), 1.97–2.08 (m, 4H), 2.27 (d, J = 14 Hz, 8H), 2.44 (s, 6H), 2.56 (s, 6H), 2.93 (t, J = 8 Hz, 2H), 3.08 (t, J = 7.6 Hz, 2H), 3.19 (m, 6H), 3.66 (t, J = 7.6 Hz, 1H), 6.00 (t, 1H), 6.92 (d, J = 7.6 Hz, 4H), 6.98 (d, J 7.2 Hz, 4H), 7.12–7.20 (m, 5H). ¹³C NMR (400 MHz CDCl₃) δ 14.42, 21.17, 21.20, 21.29, 22.93, 23.01, 24.46, 24.72, 24.82, 25.68, 27.10, 28.91, 28.98, 29.65, 29.92, 30.53, 32.74, 34.45, 36.46, 36.66, 38.74, 43.35, 44.09, 45.13, 45.29, 45.78, 48.86, 60.66, 125.74, 126.65, 127.65, 128.80, 132.32, 133.33, 133.36, 140.24, 142.63, 142.82, 143.83, 171.48, 173.85. IR (cm⁻¹) 3367.2, 2931.4, 172.0, 1645.0, 1593.1, 1541.0.

15-{N-[2,4,6-(Trimethyl)benzenesulfonyl-N-[3,3-(diphenyl)propyl]}amino-11-[N-2,4,6-(trimethyl)-benzenesulfonyl]-6-oxo-7,11-diazapentadecanoic Acid (36d)

Compound 36d was synthesized from 35d using the method described for the synthesis of 30d in 80% yield. ¹H NMR (400 MHz CDCl₃) δ 1.41 (m, 4H), 1.43–1.68 (m, 6H), 1.99 (m, 2H), 2.11 (t, J = 6.8 Hz, 2H), 2.27 (d, J = 17.2 Hz, 6H), 2.44 (s, 6H), 2.58 (s, 6H), 2.93 (t, J = 8 Hz, 2H), 3.09 (t, J = 6.8 Hz, 2H), 3.20 (m, 6H), 3.66 (t, J = 7.6 Hz, 1H), 6.08 (s, 1H), 6.93 (d, J = 8 Hz, 4H), 6.98 (d, J = 6.8 Hz, 4H), 7.12–7.21 (m, 5H). ¹³C NMR (400 MHz CDCl₃) δ 14.40, 21.16, 21.19, 21.27, 22.92, 23.08, 24.45, 24.71, 25.14, 27.098, 29.91, 32.72, 33.78, 36.22, 36.54, 43.42, 44.06, 45.18, 45.28, 48.84, 60.64, 126.64, 127.63, 128.78, 132.26, 132.31, 133.30, 140.24, 142.65, 142.81, 143.81, 171.45, 173.37, 177.76. IR (cm⁻¹) 3364.3, 2936.2, 1723.2, 1710.3, 1632.4, 1600.0, 1314.6, 1152.4.

15-{N-[2,4,6-(Trimethyl)benzenesulfonyl-N-[(4-tert-butyl)benzyl]}amino-11-[N-2,4,6-(trimethyl)benzene-sulfonyl]-6-oxo-7,11-diazapentadecanohydroxamic Acid (37a)

Compound 37a was synthesized from 36a according to the method described for the synthesis of 31d in 76.6% yield. ¹H NMR (CDCl₃) δ 1.27 (broad s, 13H), 1.61 (m, 4H), 2.16 (broad s, 2H), 2.3 (m, 8H), 2.54 (s, 6H), 2.62 (s, 8H), 2.99 (m, 4H), 3.24 (m, 4H), 4.11 (s, 2H), 6.87 (d, J = 6.8 Hz,2H), 6.95 (s, 4H) 7.28 (d, J = 7.2 Hz, 2H). ¹³C NMR (400 MHz CDCl₃) δ 21.18, 23.10, 24.14, 24.40, 24.88, 27.28, 31.50, 34.71, 35.88, 36.83, 43.52, 44.58, 45.15, 48.89, 50.89, 125.72, 128.37, 132.30, 132.52, 133.23, 133.30, 140.23, 140.28, 142.85, 151.09, 174.16. IR (cm⁻¹) 3340.5, 2936.2, 2858.4, 1716.8, 1644.5, 1600.0, 1544.2, 1457.3.

17-{N-[2,4,6-(Trimethyl)benzenesulfonyl-N-[(4-tert-butyl)benzyl]}amino-13-[N-2,4,6-(trimethyl)benzene-sulfonyl]-8-oxo-7,11-diazaheptadecanohydroxamic Acid (37b)

Compound 37b was synthesized from 36b using the method described for the synthesis of 31d in 65.9% yield. ¹H NMR (400 MHz CDCl₃) δ 1.27 (s, 9H), 1.31 (m, 8H), 1.61–1.69 (m, 4H), 2.1 (m, 3H), 2.29–2.31 (m, s, 7H), 2.58 (d, J = 7.6 Hz, 12H), 3.02 (m, 4H), 3.23 (m, 4H), 4.10 (s, 2H), 6.88 (d, J 7.6 Hz, 2H), 6.96 (s, 4H), 7.24 (s, 2H). ¹³C NMR (400 MHz CDCl₃) δ 20.97, 21.21, 23.12, 24.18, 24.46, 31.51, 34.74, 44.59, 45.21, 48.91, 125.71, 128.31, 132.34, 132.4, 132.5, 133.3, 140.26, 140.21, 142.88, 151.15, 174.12. IR (cm⁻¹) 3247.6, 2936.2, 2858.4, 1716.8, 1646.4, 1606.5, 1541.6, 1457.3.

17-{N-[2,4,6-(Trimethyl)benzenesulfonyl-N-[3,3-(diphenyl)propyl]}amino-13-[N-2,4,6-(trimethyl)-benzenesulfonyl]-8-oxo-7,11-diazaheptadecanohydroxamic Acid (37c)

Compound 37c was synthesized from 36c using the method described for the synthesis of 31d in 75.8% yield. ¹H NMR (400 MHz CDCl₃) δ 1.24–1.39 (m, 8H), 1.54–1.68 (m, 6H), 2.10 (m, 4H), 2.24 (s, 4H), 2.30 (s, 4H), 2.43 (s, 6H), 2.54 (s, 6H), 2.9 (m, 2H), 3.03 (m, 2H), 3.21 (m, 6H), 3.66 (t, 1H), 6.90 (s, 4H), 6.97 (d, J = 6.4 Hz, 4H), 7.13–7.19 (m, 6H). ¹³C NMR (400 MHz CDCl₃) δ 14.42, 21.21, 22.92, 23.10, 24.37, 24.70, 32.74, 43.42, 44.11, 45.31, 48.83, 60.65, 126.62, 127.66, 128.78, 132.26, 132.34, 133.33, 140.19, 140.24, 142.62, 142.81, 143.86, 171.45, 174.80. IR (cm⁻¹) 3254.1, 2929.7, 2858.4, 1651.9, 1632.4, 1606.5, 1554.6.

15-{N-[2,4,6-(Trimethyl)benzenesulfonyl-N-[3,3-(diphenyl)propyl]}amino-11-[N-2,4,6-(trimethyl)-benzenesulfonyl]-6-oxo-7,11-diazapentadecanohydroxamic Acid (37d)

Compound 37d was synthesized from 36d using the method described for the synthesis of 31d in 70.2% yield. ¹H NMR (400 MHz CDCl₃) δ 1.39 (m, 4H), 1.59–1.68 (m, 6H), 2.01 (m, 4H), 2.20 (s, 3H), 2.25 (s, 3H), 2.43 (s, 6H), 2.54 (s, 6H), 2.91 (m, 2H), 3.05 (m, 2H), 3.17 (m, 6H), 3.64 (t, J = 7.6 Hz, 1H), 6.90 (s, 4H), 6.98 (d, J = 7.2 Hz, 2H), 7.12–7.26 (m, 5H). ¹³C NMR (400 MHz CDCl₃) δ 14.42, 21.20, 21.29, 22.97, 23.08, 24.68, 29.93, 32.71, 44.08, 45.30, 48.83, 52.55, 60.64, 126.64, 127.65, 128.79, 132.27, 133.31, 140.23, 142.67, 143.83, 162.23, 171.44. IR (cm⁻¹) 3340.5, 1638.9, 1606.6, 1535.1, 1444.3, 1308.1.

15-N-[(4-tert-Butyl)benzyl]amino-6-oxo-7,11-diazapentadecanohydroxamic Acid dihydrobromide (12)

A 4.71 g portion of phenol (0.050 mol) was dissolved in 50 mL of 30% HBr in acetic acid in a stoppered flask, and to this mixture was added a solution of 37a (0.300 g, 0.0004 mol) in 20 mL of ethyl acetate in three portions over a period of 3 h After the addition was complete, the reaction mixture was stirred for an additional 15 h at room temperature, then cooled to 0 °C, and diluted with 100 mL of water. The aqueous phase was washed with two 100 mL portions of ethyl acetate before being lyophylized to give the crude product as yellow solid. This crude product was washed with methanol and filtered to yield the tetrahydrobromide salt of 12 (0.186 g, 77.8%) as an off white solid. An analytical sample of 12 was prepared by recrystallization from aqueous ethanol. ¹H NMR (D₂O) δ 1.20 (s, 9H), 1.42 (m, 4H) 1.61 (m, 4H), 2.01 (m,2H), 2.13 (m, 2H), 2.99 (m, 9H), 3.12 (m, 2H), 4.06 (s, 2H), 7.26 (d, J = 6.4 Hz, 2H), 7.24 (d, J = 6.4 Hz 2H). ¹³C NMR (400 MHz D₂O) δ 22.85, 22.92, 22.98, 23.81, 24.06, 24.52, 24.78, 25.77, 30.55, 32.13, 34.26, 35.42, 36.06, 38.93, 45.21, 46.20, 47.00, 47.08, 50.73, 126.44, 127.83, 129.94, 130.13, 153.57, 173.06, 177.18. IR (cm⁻¹) 3419.0, 2952.2, 1696.8, 1686.5, 1634.6, 1603.5, 1551.6. Anal. for C₂₄H₄₄Br₂N₄O₃: C, H, N.

17-N-[(4-tert-Butyl)benzyl]amino-8-oxo-7,11-diazaheptadecanohydroxamic Acid dihydrobromide (16)

Compound 16 was synthesized from 37b using the method described for the synthesis of 12 in 78.4% yield. An analytical sample of 16 was prepared by recrystallization from aqueous ethanol. ¹H NMR (400 MHz D₂O) 1.15–1.28 (m, 13H), 1.62 (m, 4H), 1.74 (m, 2H), 1.99 (m, 2H), 2.10 (m, 2H), 2.91 (m, 8H), 3.14 (m, 2H), 4.07 (d, J = 10.08 Hz, 2H), 7.29 (t, J = 8 Hz, 2H), 7.42 (t, J = 9.6 Hz, 2H). ¹³C NMR (400 MHz D₂O) δ 22.84, 24.48, 25.23, 25.77, 27.76, 27.91, 30.54, 32.38, 35.70, 36.00, 45.19, 46.16, 47.00, 50.71, 126.44, 127.83,129.94, 130.13, 153.44, 173.41, 177.85. IR (cm⁻¹) 3240.5, 2936.2, 1682.2, 1645.4, 1603.2, 1541.6, 1457.3. Anal. for C₂₆H₄₈Br₂N₄O₃: C, H, N.

17-N-[4-(3,3-Diphenyl)propyl]amino-8-oxo-7,11-diazaheptadecanohydroxamic Acid dihydrobromide (20)

Compound 20 was synthesized from 37c using the method described for the synthesis of 12 in 75.7% yield. An analyticalsample of 20 was prepared by recrystallization from aqueous ethanol. ¹H NMR (400 MHz D₂O) δ 1.07 (m, 4H), 1.39 (m, 4H), 1.53 (m, 4H), 1.68 (m, 2H), 1.96 (m, 2H), 2.06 (m, 2H), 2.30 (m, 2H), 2.83 (m, 8H), 3.09 (m, 2H), 3.94 (m, 1H), 7.11 (m,−1H), 7.22 (m, 8H). ¹³C NMR (400 MHz D₂O) δ 20.61, 22.76, 22.86, 24.84, 22.86, 24.84, 25.20, 25.75, 27.73, 27.89, 30.85, 32.33, 35.65, 35.93, 45.12, 45.43, 46.75, 46.94, 48.08, 61.82, 63.38, 127.15, 127.58, 129.18, 143.64, 173.58, 177.85 IR (cm⁻¹) 3390.3, 2923.2, 2845.4, 1658.4, 1632.4, 1554.6, 1444.3. Anal. for C₃₀H₄₈Br₂N₄O₃: C, H, N.

15-N-[4-(3,3-diphenyl)propyl]amino-6-oxo-7,11-diazapentadecanohydroxamic Acid dihydrobromide (21)

Compound 21 was synthesized from 37d using the method described for the synthesis of 12 in 70.1% yield. An analytical sample of 21 was prepared by recrystallization from aqueous ethanol. ¹H NMR (400 MHz D₂O) δ 1.07 (m, 4H), 1.39 (m, 4H), 1.53 (m, 4H), 1.68 (m, 2H), 1.97 (m, 2H), 2.06 (m, 2H), 2.30 (m, 2H), 2.83 (m, 8H), 3.01 (m, 2H), 3.94 (m, 2H), 7.11 (m, 1H), 7.22 (m, 8H). ¹³C NMR (400 MHz D₂O) δ 22.8, 22.89, 24.51, 24.78, 25.79, 30.89, 35.42, 36.02, 45.12, 45.18, 46.49, 46.72, 46.81, 46.97, 48.13, 127.18, 127.18, 127.62, 129.22, 143.68, 177.23. IR (cm⁻¹) 3383.8, 2903.8, 1658.4, 1632.4, 1561.1, 1444.3. Anal. for C₂₈H₄₄Br₂N₄O₃: C, H, N.

1-(Phthalimido)-4,8,12-tris{N-[2,4,6-(trimethyl)benzenesulfonyl]}-15-N-[2,4,6-trimethylbenzenesulfonyl)-amino]-4,8,12-triazapentadecane (39)

Compound 39 was synthesized from 38 using the procedure described for the synthesis of 33a in 65.7% yield. ¹H NMR (CDCl₃) δ 1.22 (m, 4H), 1.33–1.44 (m, 2H), 1.61–1.72 (m, 2H), 2.22 (s, 12H), 2.53 (s, 12H), 2.58 (s, 12H), 2.8 (q, 2H), 2.95 (m, 8H), 3.2 (t, J = 7.2 Hz, 2H), 6.99 (d, J = 6.8 Hz, 8H), 7.73 (m, 2H), 7.83–7.85 (m, 2H).

1-(Phthalimido)-4,8,12-tris{N-[2,4,6-(trimethyl)benzenesulfonyl]}-15-{N-[2,4,6-(trimethyl)benzenesulfonyl]-N-[2-(phenyl)benzyl]}amino-4,8,12-triazapentadecane (40a)

1-(Phthalimido)-4,8,12-tris{N-[2,4,6-(trimethyl)benzenesulfonyl]}-15-{N-[2,4,6-(trimethyl)benzenesulfonyl]-N-[(cyclopropyl)methyl]}amino-4,8,12-triazapentadecane (40b)

1-(Phthalimido)-4,8,12-tris{N-[2,4,6-(trimethyl)benzenesulfonyl]}-15-{N-[2,4,6-(trimethyl)benzenesulfonyl]-N-[(cycloheptyl)methyl]}amino-4,8,12-triazapentadecane (40c)

Compounds 40a–c were synthesized from 39 and the appropriate alkyl halide using the method described for the synthesis of 33a

Compound 40a (65% yield). ¹H NMR (400 MHz CDCl₃) δ 1.08 (m, 2H), 1.48(m, 2H), 1.62(m, 4H), 2.21(m, 12H), 2.43(s, 6H), 2.46(s, 6H), 2.52(m, 14H), 2.64(t, J = 6.8 Hz, 2H), 2.75(t, J = 7.2 Hz, 2H), 2.91–3.07(m, 8H), 3.42(t, J = 6.8 Hz, 2H), 4.27(s, 2H), 6.77(s, 2H), 6.85(s, 2H), 6.91(s, 2H), 6.94(s, 2H), 7.16(m, 3H), 7.26(m, 3H), 7.38(m, 3H), 7.72(m, 2H), 7.80(m, 2H).

Compound 40b (73% yield). ¹H NMR (CDCl₃) δ 0.02 (m, 2H), 0.45 (dd, J = 8 Hz, 5.2 Hz, 2H), 0.75 (m, 1H), 1.66 (m, 8H), 2.18 (s, 2H), 2.25 (s, 2H), 2.30 (s, 8H), 2.48 (s, 6H), 2.56 (s, 18H), 2.91 (d, J = 6.8 Hz, 2H), 3.01 (m, 12H), 3.15 (t, J = 7.2 Hz, 2H), 3.44(t, J = 6.8 Hz, 2H), 6.79 (s, 2H), 6.94 (s, 6H), 7.76 (m, 2H), 7.82 (m, 2H).

Compound 40c (72.2% yield). ¹H NMR (400 MHz CDCl₃) δ 0.77 (m,2H),1.22 (m, 8H), 1.33–1.44(m, 6H), 1.61–1.72 (m, 6H), 2.22 (s, 12H), 2.53 (s, 12H), 2.58 (s,12H),2.7 (d, J = 6.8 Hz, 2H) 2.8 (q, J = 6.6 Hz, 2H),2.95 (m, 12H), 3.2 (t, J = 7.2 Hz, 2H), 6.99 (d, J = 7.6 Hz, 8H), 7.73 (m, 2H), 7.83–7.85 (m, 2H).

11,15,19-Tris{N-[2,4,6-(trimethyl)benzenesulfonyl]}-22-{N-[2,4,6-trimethylbenzenesulfonyl)]-N-[2-(phenyl)-benzyl]amino}-6-oxo-7,11,15,19-tetraazadocosanoic Acid Methyl Ester (41a)

11,15,19-Tris{N-[2,4,6-(trimethyl)benzenesulfonyl]}-22-{N-[2,4,6-trimethylbenzenesulfonyl)]-N-[(cyclopropyl)-methyl]amino}-6-oxo-7,11,15,19-tetraazadocosanoic Acid Methyl Ester (41b)

11,15,19-Tris{N-[2,4,6-(trimethyl)benzenesulfonyl]}-22-{N-[2,4,6-trimethylbenzenesulfonyl)]-N-[2-(cycloheptyl)-methyl]amino}-6-oxo-7,11,15,19-tetraazadocosanoic Acid Methyl Ester (41c)

Compounds 41a–c were synthesized from 40a–c and 28a in two steps using the methods described for the synthesis of 27a and 29a.

Compound 41a (70% yield). ¹H NMR (CDCl₃) δ 1.08 (m, 2H), 1.48 (m, 2H), 1.43–1.79 (m, 8H), 2.05 (m, 2H), 2.21–2.39 (m, 14H), 2.53 (m, 28H), 2.77 (t, J = 7.2 Hz, 2H), 2.98 (m, 8H), 3.12 (m, 4H), 3.61 (s, 3H), 4.19 (s, 2H), 6.91 (s, 2H), 6.94 (m, 6H), 7.13 (m, 4H), 7.24 (m, 3H), 7.35 (m, 2H). ¹³C NMR (400 MHz CDCl₃) δ 21.18, 22.98, 23.02, 23.05, 23.08, 24.26, 24.68, 25.11, 25.28, 25.41, 25.78, 27.13, 33.91, 34.02, 36.25, 42.75, 43.25, 43.28, 46.18, 51.75, 109.99, 127.69, 127.78, 128.98, 128.65, 129.19, 129.46, 130.29, 132.18, 132.29, 132.34, 132.85, 133.06, 140.06, 140.26, 140.44, 142.46, 142.80, 142.89, 142.95, 172.45, 174.32. IR (cm⁻¹) 3336.0, 2931.4, 1740.5, 1655.3, 1582.7.

Compound 41b (74% yield). ¹H NMR (CDCl₃) δ 0.01 (m, 2H), 0.46 (m, 2H), 0.75 (m, 1H), 1.58 (m, 12H), 2.01 (m, 2H), 2.32 (s, 12H), 2.43 (m, 2H), 2.68 (s, 24H), 2.92 (d, J = 6.8 Hz, 2H), 3.06 (m, 10H), 3.18 (m, 4H), 3.78 (s, 3H), 6.98 (m, 8H). IR (cm⁻¹) 3383.7, 1742.7, 1664.8, 1606.4, 1561.0.

Compound 41c (70.2% yield). ¹H NMR (CDCl₃) δ 0.77 (m, 2H), 1.22 (m, 2H), 1.33–1.44 (m, 8H), 1.61–1.78 (m, 12H), 2.1 (t, 2H), 2.22 (m, 14H), 2.53 (s, 12H), 2.58 (s, 14H), 2.75 (d, J = 6.8 Hz, 2H), 2.9–3.01 (m, 12H), 3.14–3.18 (m, 4H), 3.67 (s,−3H), 6.88–6.94 (m, 8H). ¹³C NMR (400 MHz CDCl₃) δ 21.19, 23.06, 24.32, 24.49, 24.66, 25.29, 25.39, 25.95, 27.11, 28.70, 31.52, 33.78, 33.87, 36.28, 36.40, 36.47, 43.45, 43.52, 43.64, 51.76, 51.89, 132.15, 132.30, 132.34, 133.18, 140.15, 140.27, 142.95, 173.24, 174.02, 174.17, 178.84.

11,15,19-Tris{N-[2,4,6-(trimethyl)benzenesulfonyl]}-22-{N-[2,4,6-trimethylbenzenesulfonyl)]-N-[2-(phenyl)benzyl]amino}-6-oxo-7,11,15,19-tetraazadocosanohydroxamic Acid (42a)

11,15,19-Tris{N-[2,4,6-(trimethyl)benzenesulfonyl]}-22-{N-[2,4,6-trimethylbenzenesulfonyl)]-N-[(cyclopropyl)-methyl]amino}-6-oxo-7,11,15,19-tetraazadocosanohydroxamic Acid (42b)

11,15,19-Tris{N-[2,4,6-(trimethyl)benzenesulfonyl]}-22-{N-[2,4,6-trimethylbenzenesulfonyl)]-N-[2-(cycloheptyl)-methyl]amino}-6-oxo-7,11,15,19-tetraazadocosanohydroxamic Acid (42c)

Compounds 42a–c were synthesized from 41a–c in two steps using the methods described for the synthesis of 30a and 31a.

Compound 42a (62.6% yield). ¹H NMR (CDCl₃) δ 1.41–1.79 (m, 12H), 2.13 (m, 2H), 2.21–2.38 (m, 14H), 2.40–2.59 (m, 28H), 2.78(t, J = 7.2 Hz, 2H), 2.98(m, 6H), 3.18(m, 2H), 4.20–(s, 2H), 6.88(s, 2H),6.96(m, 6H), 7.15–7.43(m, 9H) ¹³C NMR (400 MHz CDCl₃) δ 14.42, 21.13, 21.18, 21.21, 21.28, 23.02, 23.09, 24.15, 24.40, 27.61, 31.61, 39.57, 42.39, 43.25, 43.82, 45.24, 46.15, 51.71, 60.61, 110.12, 127.66, 127.77, 127.99, 128.64, 129.19, 129.48, 130.27, 132.14, 132.33, 133.08, 133.31, 134.19, 139.11, 140.03, 140.17, 140.29, 140.45, 142.12, 142.46, 142.62, 142.83, 142.93, 168.21, 174.13 IR (cm⁻¹) 3407.6, 2910.1, 2765.4, 1664.7, 1644.9, 1603.6, 1447.84.

Compound 42b (60.9% yield). ¹H NMR (CDCl₃) δ 0.01 (m, 2H), 0.5 (m, 2H), 0.72 (m, 1H), 1.73 (m, 12H), 2.29 (m, 2H), 2.30 (m, 2H), 2.41 (s, 12H), 2.65 (s, 24H), 2.88 (d, J = 6.8 Hz, 2H), 3.04 (t, J = 7.6 Hz, 2H), 3.10 (m, 8H), 3.21 (t, J = 7.2 Hz, 2H), 3.28 (m, 4H), 7.06 (s, 8H). ¹³C NMR (400 MHz CDCl₃) δ 5.05, 11.08, 17.20, 18.02, 18.96, 19.05, 19.09, 20.71, 21.30, 21.55, 25.94, 31.74, 32.63, 46.13, 128.21, 128.31, 128.35, 128.96, 129.07, 129.21, 136.29, 138.75, 139.0, 150.92, 169.62, 172.23. IR (cm⁻¹) 3312.0, 2904.4, 1658.2, 1645.4, 1605.8, 1540.9.

Compound 42c (75.9% yield). ¹H NMR (CDCl₃) δ 0.77 (m, 4H), 1.33–1.44 (m, 8H), 1.61–1.78 (m, 12H), 2.15–2.27 (m, 4H), 2.22 (s, 12H), 2.53 (s, 12H), 2.58 (s,12H), 2.66 (d, J = 7.2 Hz, 2H), 2.9–3.01 (m, 12H), 3.14–3.18 (m, 4H), 6.92–6.98 (m, 8H).¹³C NMR (400 MHz CDCl₃) δ 14.42, 21.19, 21.29, 23.02, 23.06, 24.72, 24.88, 25.36, 25.94, 28.71, 36.44, 43.38,43.56, 60.64, 64.60, 132.15, 132.34, 132.98, 133.10, 133.33, 133.97, 140.14, 140.26, 142.70, 142.97, 171.44, 173.82.

22-{N-[2-(Phenyl)benzyl]amino}-6-oxo-7,11,15,19-tetraazadocosanohydroxamic Acid (7)

Compound 7 was synthesized from 42a using the method described for the synthesis of 12 in 68% yield. ¹H NMR (D₂O) δ 1.48 (m, 4H), 1.79 (m, 4H), 1.98 (m, 4H), 2.15 (m, 2H), 2.23 (m, 2H), 2.75 (t, J = 8.0 Hz, 2H), 2.8–3.05 (m, 12H), 3.12 (t, J = 6.4 Hz, 2H), 4.16 (s, 2H), 7.24–7.44 (m, 9H). ¹³C NMR (400 MHz D₂O) δ 22.42, 22.76, 22.83, 23.75, 23.81, 24.83, 25.77, 33.42, 33.51, 35.47, 36.06, 43.92, 44.62, 44.67, 45.44, 48.23, 115.47, 120.78, 128.05, 128.21, 128.67, 129.10, 129.56, 129.90, 130.08, 130.12, 130.97, 139.53, 142.81, 177.23, 178.65. IR (cm⁻¹) 3419.0, 3045.5, 2910.6, 1688.5, 1651.3, 1603.4. Anal. for C₃₁H₅₄Br₄N₆O₃: C, H, N.

22-{N-[(Cyclopropyl)methyl]amino}-6-oxo-7,11,15,19-tetraazadocosanohydroxamic Acid (9)

Compound 9 was synthesized from 42b using the method described for the synthesis of 12 in 65% yield. ¹H NMR (D₂O) δ 0.22 (m, 2H), 0.54 (m, 2H), 0.91 (m, 1H), 1.45(m, 5H), 1.75 (q, J = 6.8 Hz, 3H), 1.98 (m, 4H), 2.13 (t, J = 6.8 Hz, 2H) 2.25(t, J = 7.2 Hz, 2H), 2.83 (d, J = 7.6 Hz, 2H), 2.93 (t, J = 7.6 Hz, 2H), 3.05 (m, 12H), 3.14 (t, J = 7.2 Hz, 2H). IR (cm⁻¹) 3312.1, 2931.4, 1655.3, 1645.4, 1593.0. Anal. for C₂₂H₅₀Br₄N₆O₃: C, H, N.

22-{N-[2-(Cycloheptyl)methyl]amino}-6-oxo-7,11,15,19-tetraazadocosanohydroxamic Acid (10)

Compound 10 was synthesized from 42c using the method described for the synthesis of 12 in 70.4% yield ¹H NMR (D₂O) δ 1.08–1.16 (m, 4H), 1.28–1.58(m, 14H), 1.73–1.78 (m, 2H), 1.93–2.07 (m, 6H), 2.78 (d, J = 8.8 Hz, 2H), 2.93 (t, J = 7.2 Hz, 2H) 2.96–3.01 (m, 12H), 3.12–3.16 (t, J = 6.8 Hz, 2H).¹³C NMR (400 MHz D₂O) δ 22.66, 22.79, 24.52, 24.76, 25.42, 25.81, 27.95, 31.25, 32.12, 35.42, 36.01, 36.30, 173.11, 177.26 IR (cm⁻¹) 3408.7, 2910.7, 2848.4, 1665.7, 1644.0, 1447.8. Anal. for C₂₆H₅₈Br₄N₆O₃: C, H, N.

7-{4-[(Phthalimido)amino]butyl}-{N-[4-N,N-(dimethyl)-aminobenzyl]carbamoyl}heptanoic Acid Methyl Ester (43)

Compound 43 was synthesized from 25c and 28b using the procedure described for the synthesis of 29c in 72.6% yield. ¹H NMR (400 MHz CDCl₃) δ 1.24–1.35 (m, 4H), 1.61–1.64 (m, 8H), 2.26–2.36 (m, 4H), 2.92 (d, J = 8.8 Hz, 6H), 3.20 (t, J = 7.2 Hz, 1H), 3.36 (t, J = 6.8 Hz, 1H), 3.65–3.71 (m, 5H), 4.42 (s, 1H), 4.48 (s, 1H), 6.65 (d, J = 8.8 Hz, 1H), 6.69 (d, J = 8.4 Hz, 1H), 7.01 (d, J = 7.6 Hz, 1H), 7.11 (d, J = 8.4 Hz, 1H), 7.69–7.75 (m, 2H), 7.80–7.86 (m, 2H). ¹³C NMR (400 MHz CDCl₃) δ 24.82, 24.99, 25.56, 26.21, 28.92, 28.97, 29.20, 29.29, 33.24, 33.40, 34.19, 37.57, 37.86, 40.81, 40.86, 45.39, 46.23, 47.62, 50.79, 51.65, 112.84, 113.00, 123.37, 123.50, 124.50, 125.84, 127.47, 129.46, 132.19, 134.08, 134.26, 150.11, 150.21, 168.57, 173.08, 174.45 IR (cm⁻¹) 3461.6, 2936.2, 2858.4, 1742.7, 1768.7, 1710.3, 1638.9, 1613.0, 1515.7.

7-{4-[N-(tert-Butyloxycarbonyl)amino]butyl}-{N-[4-N,N-(dimethyl)aminobenzyl]carbamoyl}heptanoic Acid (44)

Compound 44 was synthesized from 43 in three steps using the procedures described for the synthesis of 27b (phthalimide cleavage) and 26c (N-Boc protection) and 31c in 70.2% overall yield. ¹H NMR (400 MHz CDCl₃) δ 1.25 (m, 2H), 1.30 (m, 2H), 1.39 (s, 12H), 1.47 (m, 2H), 1.58 (m, 4H), 2.15 (m, 2H), 2.29 (m, 2H), 2.86 (d, J = 7.2, 6H), 3.03 (m, 2H), 3.11 (t, J = 7.6, 1H), 3.26 (t, J = 7.2 Hz, 1H), 4.35 (s, 1H), 4.42 (s, 1H), 4.85 (s, 1H), 6.60 (d, J = 8.8 Hz, 1H), 6.64 (d, J = 8.8 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 7.04 (d, J = 8.8 Hz, 1H). ¹³C NMR (400 MHz CDCl₃) δ 14.32, 21.24, 24.24, 25.28, 25.36, 25.86, 27.54, 28.36, 28.62, 28.95, 33.30, 33.94, 40.25, 40.39, 40.84, 40.89, 45.56, 46.47, 47.63, 50.77, 60.60, 81.44, 112.90, 113.09, 124.51, 127.49, 129.38, 150.08, 150.25, 156.30, 171.42, 173.24, 173.32, 173.70. IR (cm⁻¹) 3280.0, 2975.1, 2929.7, 2864.9, 1690.8, 1619.5, 1522.2.

7-{4-[N-(tert-Butyloxycarbonyl)amino]butyl}-{N-[4-N,N-(dimethyl)aminobenzyl]carbamoyl}heptano-hydroxamic Acid (45)

Compound 45 was synthesized from 44 using the procedure described for the synthesis of 31c in 74.4% yield. ¹H NMR (400 MHz CDCl₃) δ 1.31 (m, 4H), 1.37 (m, 4H), 1.64 (m, 4h), 2.29–2.36 (m, 4H), 2.94 (d, J = 8.4 Hz, 6H), 3.09 (m, 2H), 3.27 (t, J = 7.2 Hz, 1H), 3.32 (t, J = 8.6 Hz, 1H), 4.41 (s, 1H), 4.48 (s, 1H), 6.66 (d, J = 8.4 Hz, 1H), 6.72 (d, J = 8.8 Hz, 1H), 7.02 (d, J = 8.8 Hz, 1H), 7.1 (d, J = 8.8 Hz, 1H), 8.22 (d, J = 8.8 Hz, NH). ¹³C NMR (400 MHz CDCl₃) δ 25.00, 25.26, 25.35, 25.88, 27.60, 28.37, 28.64, 28.92, 29.90, 33.00, 33.27, 34.01, 40.28, 40.44, 40.87, 40.91, 45.58, 46.47, 47.65, 50.79, 81.63, 112.89, 113.10, 124.55, 127.50, 129.44, 150.12, 150.27, 155.91, 156.27, 173.19, 173.67, 174.97. IR (cm⁻¹) 3267.0, 2975.1, 2936.2, 2864.9, 1684.3, 1677.8, 1619.5, 1509.2, 1450.8.

7-{[4-(Amino)butyl]-N-[4-(dimethylaminobenzyl)-carbamoyl]}heptanohydroxamic Acid (19)

synthesized from 32 according to the procedure mentioned for 13 in 90.9% yield. ¹H NMR (400 MHz CD₃OD) δ 1.31–1.40 (m, 4H), 1.64 (m, 8H), 2.26–2.36 (m, 2H), 2.38 (t, J = 4.8 Hz, 1H), 2.49 (t, J = 4.8 Hz, 1H), 2.94 (m, 2H), 3.29 (s, 6H), 3.39 (m, 2H), 4.65 (s, 1H), 4.73 (s, 1H), 7.45 (t, J = 7.2 Hz, 2H), 7.62 (d, J = 6 Hz, 1H), 7.67 (d, J = 5.6 Hz, 1H). ¹³C NMR (400 MHz CD₃-OD) δ 24.14, 24.59, 24.72, 24.77, 24.97, 25.13, 25.42, 28.62, 28.68, 28.78, 32.52, 32.86, 39.11, 50.33, 114.58, 117.45, 120.59, 120.94, 128.400, 129.46, 139.73, 140.57, 141.98, 160.22, 173.05, 174.63, 175.03. IR (cm⁻¹) 2936.2, 1671.4, 1626.0, 1515.7, 1463.8, 1424.8. Anal. for C₂₃H₃₇F₃N₄O₅: C, H, N.

1-N-[2,4,6-(Trimethyl)benzenesulfonyl]amino-4-N-[2,4,6-(trimethyl)benzenesulfonyl]-8-N-{4-[N,N-(dimethyl)-benzyl]amino}-4-azaoctane (46)

Compound 46 was synthesized in two steps from 32 using the procedure described for the syntheses of 27c and 25c in 77.3% overall yield. ¹H NMR (400 MHz CDCl₃) δ 1.35 (q, J = 7.6 Hz, 2H), 1.47 (q, J = 6.8 Hz, 2H), 1.57 (q, J = 6.8 Hz, 2H), 2.28 (d, J = 2.8 Hz, 6H), 2.51–2.59 (m, 14H), 2.78 (t, J = 6.8 Hz, 2H), 3.13 (t, J = 7.6 Hz, 2H), 3.17 (t, J = 7.6 Hz, 2H), 6.93 (d, J = 10 Hz, 4H). ¹³C NMR (400 MHz CDCl₃) δ 14.21,19.08, 22.47, 25.6, 26.22, 27.46, 32.41, 40.12, 40.1, 43.88, 46.18, 127.44, 128.14, 136.25, 137.49, 141.58, 142.33.

5-[(4-N,N-(Dimethylamino)benzyl)–(4-{(2,4,6-trimethyl-benzenesulfonyl)-[3-(2,4,6-trimethyl-benzenesulfonylamino)propyl]amino}butyl)carbamoyl]pentanoic Acid Methyl Ester (47)

Compound 47 was synthesized from 46 and 28a using the procedure described for the synthesis of 29d in 81.6% yield. ¹H NMR (400 MHz CDCl₃) δ 1.26 (m, 2H), 1.63 (m, 2H), 1.68 (m, 6H), 2.29 (m, 2H), 2.32 (m, 6H), 2.36 (m, 2H), 2.52 (m, 6H), 2.62 (m, 6H), 2.79 (m, 2H), 2.92 (s, 6H), 3.01–3.13 (m, 6H), 3.67 (s, 3H), 4.29 (s, 1H), 4.35 (s, 1H), 6.68 (d, J = 8.4 Hz, 2H), 6.88–6.95 (m, 6H). IR (cm⁻¹) 3409.7, 2923.2, 1736.8, 1664.8, 1619.4, 1528.6.

5-[(4-N,N-(Dimethylamino)benzyl)–(4-{(2,4,6-trimethyl-benzenesulfonyl)-[3-(2,4,6-trimethyl-benzenesulfonylamino)propyl]amino}butyl)carbamoyl]pentanohydroxamic Acid (48)

Compound 48 was synthesized in two steps from 47 using the procedures described for the syntheses of 30d and 31d in 66.2% overall yield. ¹H NMR (400 MHz D₂O) δ 1.40 (m, 4H), 1.50 (m, 2H) 1.63 (m, 4H) 1.80 (m, 1H), 1.98 (m, 1H), 2.07 (m, 1H), 2.31 (m, 1H), 2.71 (m, 1H), 2.90–2.93 (m, 8H), 3.14 (d, J = 4.4 Hz, 6H), 3.38 (m, 2H), 7.31 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.8 Hz, 1H), 7.49 (d, J = 8.8 Hz, 1H). IR (cm⁻¹) 3398.3, 2941.8, 2683.2, 1613.0, 1522.2, 1470.3, 1184.9, 990.3.

5-[(4-N,N-(Dimethylamino)benzyl)–(4-{(2,4,6-trimethyl-benzenesulfonyl)-[3-(2,4,6-trimethyl-benzenesulfonylamino)propyl]amino}butyl)carbamoyl]pentano-hydroxamic Acid (15)

Compound 15 was synthesized in two steps from 48 using the procedure described for the synthesis of 12 in 82.2% yield. ¹H NMR (400 MHz D₂O) δ 1.14 (s, 9H), 1.43 (m, 4H), 1.59 (m, 4H), 1.72 (q, J = 7.6 Hz, 2H), 2.11 (t, J = 6.4 Hz, 2H), 2.32 (t, J = 7.2 Hz, 2H), 2.85–2.94 (m, 6H), 3.12 (t, J = 6.4 Hz, 2H), 4.05 (s, 2H), 7.27 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 1.6 Hz, 2H). ¹³C NMR (400 MHz D₂O) δ 22.83, 22.96, 23.80, 24.05, 24.84, 25.77, 30.53, 33.49, 34.26, 35.43, 36.02, 45.17, 46.15, 46.97, 50.17, 126.44, 127.81, 129.92, 153.57, 177.28, 178.68. IR (cm⁻¹) 3396.8, 2929.7, 1677.8, 1606.5, 1424.9.

Histone Deacetylase Activity Assay

Compounds 6–21 were evaluated for their ability to inhibit isolated HDAC in a commercially available assay (Fluor de Lys Assay System, Biomol International LP, Plymouth Meeting, PA), employing 1 and 2 as positive controls. The reaction mixture contains a HeLa cell nuclear extract and a commercial substrate containing acetylated lysine side chains. The substrate and extract are incubated in the presence of the appropriate concentration of the inhibitor. Deacetylation of the substrate followed by mixing with mixing with the provided developer generates a fluorophore, and comparison of inhibited vs control relative fluorescence using a standard plate reader was employed to determine percent HDAC activity remaining. All determinations were carried out in triplicate, and reported values are the average of these determinations, which in no case varied by more than 3%.

Cell Lines and Drug Treatment

ML-1 cells were maintained in RPMI medium supplemented with 10% fetal calf serum, 0.1 mg/mL gentamicin, and 2 mM l-glutamine. 3 × 10⁵ cells/ml were treated with 1 (Wako Pure Chemicals, Richmond, VA), 2 (Mitsui Pharmaceuticals, Chiba, Japan) and the desired test compound for the concentration and time indicated in the figure legend.

Histone Preparation

Histones were prepared by a modification of a previously described method.²⁴ Cells were washed in 2 mL of HBSS and disrupted by 1 mL of ice-cold lysis buffer A (10 mM Tris pH 7.6, 5 mM butyric acid, 1% Triton X-100, 1 mM MgCl₂, and 1 mM PMSF). Nuclei were collected by centrifugation at 14 000 rpm for 15 min. The pellet was resuspended once with 250 μL of ice-cold lysis buffer B (10 mM Tris pH 7.6, 0.25 M Sucrose, 3 mM CaCl₂, and 5 mM butyric acid). Sulfuric acid was added to a concentration of 0.4 N, and the tubes were incubated at 4 °C for overnight. Debris was pelleted by centrifugation, and the supernatant was collected. Histones were precipitated by addition of 10 vol of acetone and incubated at −20 °C overnight. Pellets were collected by centrifugation, briefly dried under vacuum, and resuspended in ddH₂O.

p21^WAF1/Cip1 Expression Analysis

ML-1 cells after treatment were lysed in RIPA lysis buffer containing an EDTA-free protease inhibitor cocktail, at 4 °C for 30 min. Lysate was clarified by centrifugation at 14 000 rpm for 15 min. The resulting supernatant was used for analysis. The total protein content was determined by a bicinchoninic acid (BCA) assay kit (Pierce, Rockford, IL), and the absorbance of the solution was measured using a spectrophotometer at a wavelength of 570 nm. Absorbance was converted to protein content using an albumin standard curve.

The proteins (10 μg for histone or 30 μg for p21^Waf1) were separated by 15% SDS PAGE and visualized by Western blot analysis using the following antibodies against interesting proteins: antibodies for acetylhistone H3 (06-599) (diluted 1:1000), acetylhistone H4 (06-866) (diluted 1:500), and histone H2A (07-146) (diluted 1:1000) were from Upstate Biotechnologies, p21^Waf1 (556431) (diluted 1:500) from BD Pharmigen, and β-actin (ON365) (diluted 1:1000) from Oncogene Research Products. The immunoreactive proteins were detected using ECL western blotting analysis system (Amersham Biosciences, Piscataway, NJ). Cell proliferation was quantified by the MTT assay according to the supplied protocol (Promega, Madison, WI).