Histone deacetylase

Synthesis of Selective Inhibitors of Histone Deacetylase

Abstract

Histone deacetylase (HDAC) enzymes are essential to the regulation of gene expression but overexpression has been linked to cancer formation. Compounds which inhibit HDAC activity are thus potential anti-cancer drugs.

Chlamydocin-benzamide analogs were synthesised as potential HDAC inhibitors based on the structures of chlamydocin and MS-275. Chlamydocin is a cyclic tetrapeptide containing an epoxyketone moiety as the zinc-binding ligand that makes it an irreversible HDAC inhibitor. Benzamide MS-275 acts as a reversible, but less potent inhibitor of HDAC. Both chlamydocin and MS-275 exhibit selectivity for Class I HDACs. In this project, the chlamydocin tetrapeptide capping framework was combined with the benzamide metal-binding moiety of MS-275 to design potentially potent, reversible and selective HDAC inhibitors.

The successful synthesis of a chlamydocin-benzamide amide analog via a convergent approach is reported here. This analog will be screened for HDAC inhibitory activity to determine its potency and selectivity profile.

The challenges faced in attempting to synthesise the corresponding chlamydocin-benzamide amine analog are also described in this report. The main issue is the undesired cyclisation of an aldehyde intermediate onto an amide nitrogen atom of the tetrapeptide into its γ-hydroxylactam form, which is relatively unreactive. Alternatives to overcome this are proposed.

1. Introduction

1.1 Histone Deacetylase Enzymes

Histone deacetylase (HDAC) enzymes play a fundamental role in the regulation of gene expression. They catalyse the removal of acetyl groups from ε-amino groups of lysine amino acid residues located on the amino-terminal tails of histones during post-translational modification1.

Histone proteins associate with deoxyribonucleic acid (DNA) to form nucleosomes, the basic repeating unit of chromatin2. Deacetylation of lysine residues results in a positively charged side chain, which increases the affinity of the histone for negatively charged DNA, thus resulting in a more condensed chromatin structure1. In genomic DNA, this reduces the accessibility of genes to transcriptional proteins, and results in repression of gene transcription.

1.1.1 HDAC Isoforms

There are 18 HDAC proteins in humans, separated according to their size, sequence, active site(s) and cellular location into four classes and two families3.

Members of the classical HDAC family have similar sequences and function by a Zn2+ ion-dependent mechanism4. These can be grouped into three classes: Class I includes HDAC1, 2, 3 and 8; Class II comprises of HDAC 4, 5, 6, 7 and 9; while HDAC 11 is the only member of Class IV5. The X-ray crystal structure of human HDAC 8 complexed with inhibitor suberoylanilide hydroxamic acid (SAHA)6, as elucidated by Somoza, Skene, Katz, Mol, Ho, Jennings, Luong, Arvai, Buggy, Chi, Tang, Sang, Verner, Wynands, Leahy, Dougan, Snell, Navre, Knuth, Swanson, McRee, and Tari7,

X-ray structure of human HDAC 8 complexed with SAHA. The protein scaffold is represented in ribbon form, and the Zn2+ ion is represented by the grey sphere bound to molecular inhibitor SAHA [Somoza et al.]

The sirtuin family comprises of proteins related to silent information regulator 2 (Sir2). These Class III proteins require NAD+ for function, and do not resemble the classical HDACs in sequence8.

1.1.2 The Link to Cancer

While HDAC proteins are crucial for the control of basic cellular functions such as cell growth, differentiation and apoptosis, overexpression of HDAC is an epigenetic defect that has been linked to cancer formation9.

Although the exact function of each HDAC isoform and the mechanism of carcinogenesis have not been elucidated, studies have shown that specific Class I and Class II HDACs are overexpressed in certain cancers. For example, Class I HDACs have been associated with ovarian (HDACs 1-3)10, gastric (HDAC 2)11, and lung cancer (HDACs 1 and 3)12, while Class II HDAC has been implicated in breast cancer (HDAC 6)13.

1.2 Histone Deacetylase Inhibitors

The establishment of a connection between HDAC proteins and cancer formation has led to the development of HDAC inhibitors as potential anti-cancer therapeutic agents. In particular, inhibitors that target single isoforms or inhibitors selectively for either Class I or Class II HDACs have been identified as viable anti-cancer drugs.

To date, a wide array of compounds that inhibit HDAC activity has been found. These can be divided into 5 classes14 comprising of (i) short-chain fatty acids such as butyric acid, (ii) hydroxamic acids e.g. suberoylanilide hydroxamic acid (SAHA)6, (iii) electrophilic ketones, (iv) benzamides for instance MS-27515, and (v) natural cyclic peptides such as chlamydocin16.

Several of these compounds are in clinical trials for use as anti-cancer drugs17. In fact, HDACi suberoylanilide hydroxamic acid (SAHA)6, otherwise known as vorinostat, was approved by the US Food and Drug Administration (FDA) in 2006 for treatment of advanced cutaneous T-cell lymphoma (CTCL) and is currently marketed by Merck under the name Zolinza18.

1.2.1 Natural HDAC Inhibitors

A group of naturally occurring cyclic tetrapeptides including trapoxins19, chlamydocin16 and HC toxins20 has been found to exhibit HDAC inhibiting activity. This family of natural products shares a common structure of a four cyclically linked amino acids, including at least one amino acid with an aromatic side chain, and also 2-amino-9-oxo-9,10-epoxy decanoic acid (Aoe).

The activity of these compounds has been attributed to the long chain residue of Aoe containing the epoxyketone functionality, which enters the HDAC active site and reacts irreversibly with the functional nucleophile near the Zn atom of the catalytic site19. Still, there is evidence to indicate that the cyclic tetrapeptide moiety acts as a capping group to associate with amino acid residues at the surface of the HDAC enzyme via hydrophobic interactions21. In particular, chlamydocin displays potent anti-cancer activity in vitro, inhibiting HDAC activity with a half maximal inhibitory concentration (IC50) value of 0.15 nM22.

However, there are issues with the use of these natural products as anti-cancer therapeutics. Firstly, the majority of these compounds are pan-inhibitors of HDAC, inhibiting the activity of all human HDAC isoforms non-selectively. This is undesired as only specific HDAC enzymes have been associated with cancer formation. Secondly, compounds with reactive functionalities that confer non-specific, irreversible binding are undesirable as pharmaceuticals, since indiscriminant reactivity can lead to binding to other proteins and DNA, possibly resulting in adverse toxicological outcomes23.

1.2.2 The Quest for Isoform-Selective HDAC Inhibitors

The synthesis of selective HDAC inhibitors has been challenging due to the lack of characterisation and inhibitor docking studies on the active site of HDAC isoforms. However, a review by Bieliausakas and Pflum24 on class- and isoform- selective HDAC inhibitors in Chem. Soc. Rev. (2008) sheds light on inhibitor design.

The structure of HDAC inhibitors can be divided into 3 regions, each with a different function - the capping group, the metal-binding moiety, and the linker - and each capable of exhibiting class-selectivity.

Pflum and Bieliausakas found that HDAC inhibitors with a cyclic peptide moiety at the capping group exhibit Class I selectivity, with chlamydocin displaying selectivity for HDAC 1 (Class I) versus HDAC 6 (Class II) of 7300-fold with pM potency (HDAC 1 IC50 = 0.15 nM)24. This has been attributed to structural similarity with the substrate of Class I HDACs22.

In addition, many Class I-selective HDAC inhibitors contain benzamides (Bam) as the metal-binding moiety24. In particular, benzamide derivative entinostat (MS-275)15 exhibited at least 135-fold preference for HDACs 1 and 3 (Class I) versus HDACs 6 (Class II) and 8 (Class I). Structure-activity relationship (SAR) showed that the 2'-amino group of benzanilide was crucial for specific interaction with the HDACs, possibly by hydrogen-bonding or other electrostatic interaction25.

Furthermore, the presence of amide bonds and/or phenyl rings in the linker promotes preferential inhibition of HDACs 1 and 2 (Class I) over HDAC 3 (Class I) and HDACs 4, 5 and 6 (Class II)24. These functionalities can be observed in the linker of MS-275 as well.

1.2.3 Synthetic HDAC Inhibitors: Chlamydocin Analogs

Many compounds bearing the chlamydocin cyclic tetrapeptide framework have been synthesised as HDAC inhibitors. These include chlamydocin analogs bearing carbonyl groups26 and hydroxamic acid groups27 as metal-binding moieties. The synthesis of chlamydocin-aldehyde analogs by Bhuiyan, Kato, Okauchi, Nishino, Maeda, Nishino and Yoshida26 via solution-phase peptide coupling is highlighted in Scheme 1.

Scheme 1 Synthesis of chlamydocin-aldehyde analogs. Reagents and conditions: (a) HBTU, DIEA, Z-L-Phe, (b) Pd-C, H2, (c) HBTU, DIEA, Z-Aib, (d) HBTU, DIEA, Boc-L-Asu(OBn), (e) TFA, (f) HATU, DIEA, (g) MC-OsO4, N-methylmorpholine-N-oxide, (h) NaIO4 [Bhuiyan et al., 2006]

1.2.4 Synthetic HDAC Inhibitors: Benzamide Analogs

Benzamide derivative MS-27515 is a reversible HDAC inhibitor which displayed HDAC inhibitory activity both in vitro and in vivo (HDAC 1 IC50 = 125 nM)28, as well as a strong selectivity profile for Class I HDACs. The synthesis of MS-275 by Suzuki, Ando, Tsuchiya and Fukazawa25 is shown in Scheme 2.

Scheme 2 Synthesis of MS-275. Reagents and conditions: (a) CDI, DBU, Et3N, THF, (b) oxalyl chloride, toluene, (c) imidazole, THF, (d) benzene-1,2-diamine, TFA, THF [Suzuki et al., 1999]

1.2.5 Summary of HDAC inhibitors

The potency and selectivity of aforementioned HDAC inhibitors chlamydocin16, SAHA6 and MS-27515 are summarised in Table 122, 28. Chlamydocin16 is a highly potent HDAC inhibitor with pM potency for HDAC 1, while both SAHA6 and MS-27515 have IC50 values of the nM scale. SAHA6 is selective for HDACs 1, 2, 3, 8 (Class I) and 6 (Class II) , with IC50 values of 30 to 410 nm, over HDACS 4, 5 and 7 (Class II), where IC50 > 30,000 nM. MS-27515 has greater selectivity, with nM potency only for HDACs 1, 2 and 3 (Class I).

Table 1 HDAC selectivity of SAHA, chlamydocin and MS-275 [Furumai et al., Witter et. al.]

HDAC

IC50 / nM

Chlamydocin

SAHA

MS-275

1

0.15

30

125

2

NT

170

340

3

NT

100

370

4

NT

> 50,000

> 50,000

5

NT

> 30,000

> 50,000

6

1,100

38

> 50,000

7

NT

> 30,000

> 50,000

8

70

410

14,120

NT = not tested

1.3 Overview of Project

In this project, the synthesis of analogs comprising the chlamydocin cyclic tetrapeptide capping group and the MS-275 benzamide metal-binding moiety is proposed. Based on the above SAR, it is expected that such a combination is potentially capable of improved potency, reversibility and isoform-selectivity in the inhibition of HDAC activity.

2. Aims and Objectives

2.1 Overview of Synthetic Targets

The aim was to synthesise potent, reversible isoform-selective HDAC inhibitors by combining the chlamydocin cyclic tetrapeptide capping group and the MS-275 benzamide metal-binding moiety.

A convergent approach is adopted as it offers high overall yields and efficiency, and the option of diversification to a combinatorial library29. This is advantageous as it allows independent modification of the core fragments to adjust the potency and selectivity of the target compounds. The strategy involved the separate synthesis of the capping group and metal-binding fragments, followed by fragment coupling to form target compounds 1 and 2 (Fig 6).

As shown in Fig 6, targets 1 and 2 differ only in the linker region - target 1 has an amide linker, while in target 2 it is an amine. These linkers were chosen not only for their ease of coupling, but also because both targets can be derived from the same fragments. Thus, a common route can be employed to the synthesis of the fragments, with late-stage divergence to obtain targets 1 and 2.

2.2 Retrosynthetic Approach to Chlamydocin-Benzamide Amide Target 1

For target compound 1, benzamide 330 comprised the metal-binding fragment, while the chlamydocin analog in which Aoe was replaced by aspartic acid (Asp)31 formed the capping group fragment 4 (Scheme 3).

Scheme 3 Retrosynthetic analysis of target 1 leading to and benzamide 3 and chlamydocin-acid analog 4, and their respective precursors

As outlined in Scheme 3, retrosynthetic simplification of target 1 started with the disconnection of the amide bond linking the side chain to the tetrapeptide core, leading to benzamide 3 and chlamydocin-acid analog 4. Benzamide 330 is obtained from commercially available 4-aminomethylbenzoic acid and benzene-1,2-diamine via a series of protection, coupling and deprotection steps. chlamydocin-acid analog 4 is derived from the coupling and subsequent cyclisation of tripeptide 5, synthesised by Mr. Rob Felstead, and commercially available aspartic acid derivative Boc-Asp(OBn)-OH.

2.3 Retrosynthetic Approach to Chlamydocin-Benzamide Amine Target 2

For target compound 2, benzamide 330 again comprised the metal-binding fragment, while the capping group fragment 6 is a chlamydocin analog in which Aoe was replaced by 2-amino-4-pentenoic acid (Ae5)32 (Scheme 4).

Scheme 4 Retrosynthetic analysis of target 2 leading to benzamide 3 and chlamydocin-aldehyde analog 6, and its precursor

Scheme 4 shows the retrosynthetic simplification of target 2, beginning with disconnection of the side chain from the tetrapeptide core, leading to benzamide 330 and chlamydocin-aldehyde analog 6. chlamydocin-aldehyde analog 6 is derived from the oxidative cleavage of chlamydocin-alkene analog 7, which was synthesised by Mr. Rob Felstead.

3. Results and Discussion

3.1 Synthesis of Benzamide Fragment

The synthesis of benzamide 330 via solution-phase peptide synthesis is outlined in Scheme 5. Orthogonal33 amine protecting groups Boc and Fmoc were chosen so that amine deprotection can be achieved separately later in the synthetic sequence; Boc is acid labile and easily cleaved by TFA, while Fmoc is stable under acidic conditions and requires basic conditions for deprotection.

Scheme 5 Synthesis of benzamide 3. Reagents and conditions: (a) Fmoc-Cl (1.0 eq), Na2CO3 (3.0 eq), dioxane/ H2O (1:1), 0°C → rt, overnight; (b) Boc2O (1.0 eq), Na2CO3 (0.6 eq), NaCl (0.1 eq), DCM, 0°C → reflux, overnight; (c) HOBt (1.0 eq), DCC (1.2 eq), DIEA (1.0 eq), DMF, 0°C → rt, overnight; (d) DBU, CH2Cl2, 2 h

N-protection of 4-aminomethylbenzoic acid was achieved by reacting with Fmoc chloride to give acid 834 in 86% yield. Its 1H NMR spectrum corroborates well with literature34, except that the labile carboxylic acid proton cannot be observed due to proton exchange and hydrogen bonding. Still, the correct mass of m/z (ES-) 372.1250 ([M-H]-) was observed in the mass spectrum.

Benzene-1,2-diamine was mono-N-protected with orthogonal protecting group Boc, giving amine 935 in 67% yield [lit.35 80%]. Its spectral properties (1H NMR and MS) are a good match to literature data as well35. TLC of the reaction mixture also indicated the presence of the starting material and another by-product, possibly di-N-Boc-protected amine, which was not isolated.

For the coupling reaction, HOBt, DCC and DIEA were chosen as coupling reagents. DCC was the preferred coupling agent due to its wide usage and low price, and also for the easy removal of its coupling by-product, dicyclohexylurea, as it is insoluble in DMF36. Additive HOBt enhances the rate of coupling, and also decreases racemisation36. The reaction mechanism for a generalised DCC/HOBt/DIEA coupling of amino acids is shown in Scheme 6.

Scheme 6 Reaction mechanism for a generalized DCC/HOBt/DIEA coupling of amino acids

Coupling of acid 834 with amine 935 using DCC/HOBt/DIEA afforded amide 10 in 38% yield. The low yield can be attributed to the formation of side products during coupling. As amide 10 is not a known compound, it was identified by its spectral properties. In comparison of its 1H NMR spectrum with its precursors acid 834 and amine 935, the same peaks are observed with a slight variation in chemical shift and the loss of one N-H proton, indicating the presence of the desired product. The formula was confirmed by HRMS.

Fmoc deprotection with DBU yielded benzamide fragment 330 in 94%. Again, benzamide 330 was identified by a comparison of its NMR spectra with its precursor amide 10. The removal of the Fmoc protecting group simplified the 1H NMR spectrum. The disappearance of the signals at δ 4.45 and 4.32 ppm indicating the loss of the aliphatic protons on the Fmoc group, and appearance of a new broad signal at δ 2.07 ppm corresponding to 2 N-H protons were also observed. The 13C NMR spectrum showed a significant reduction in the number of peaks, especially in the δ 145 - 120 ppm aromatic region. Also, only 2 amide carbons remain in the δ 170 - 150 ppm region.

3.2 Synthesis of Chlamydocin-Acid Moiety

The synthesis of chlamydocin-acid analog 4 began with the coupling of tripeptide 5 with Boc-Asp(OBn)-OH, using the same coupling reagents as before, to give the protected tetrapeptide 11 in 79% yield as shown in Scheme 7. Acyclic tetrapeptide 11 experiences rotational isomerism, observable in the 1H and 13C NMR spectra. This is a result of conformational freedom allowing rotation about the bonds of the α-carbon on each amino acid residue. The relative amounts of each rotamer can be calculated from integrals in the 1H spectrum, giving a ratio of 73:27.

This was followed by Boc deprotection of both N- and C- terminals by TFA to yield the TFA salt of tetrapeptide 12 (81%). As with compound 11, acyclic tetrapeptide 12 also exists as rotamers, but in a 77:23 mixture. As the 1H NMR spectra of both acyclic tetrapeptides 11 and 12 are highly complicated by the presence of rotamers, the assignment of these spectra was carried out with reference to the 1H NMR spectrum of cyclic tetrapeptide 13.

Scheme 7 Synthesis of chlamydocin-acid analog 4. Reagents and conditions: (a) HOBt (1.0 eq), DCC (1.2 eq), DIEA (1.0 eq), DMF, 0°C → rt, overnight; (b) TFA, 0°C, 2 h; (c) HBTU (1.2 eq), DIEA (3.2 eq), DMF, overnight; (d) H2, Pd-C, MeOH, 3 days

Cyclisation of the tetrapeptide was achieved by reaction with HBTU and DIEA under high dilution conditions in DMF, following the procedure of Bhuiyan et al.26 HBTU was chosen instead of HATU, which was utilised by Bhuiyan et al., due to its lower cost36. Under these conditions, protected cyclic tetrapeptide 13 was obtained in 47% yield. Since rotation in the cyclic tetrapeptide is constrained by the ring, it does not have rotamers unlike its acyclic counterparts. As a result, its NMR spectra are cleaner than that of its precursors; and its 1H NMR spectrum is the basis for assignment of the spectra of compounds 11 and 12. Key 1H NMR evidence for the formation of 13 include (i) 2 sets of singlets with 3 protons each at δ 1.30 and 1.72 ppm corresponding to Aib, (ii) multiplets from methine protons on the α-carbons of the amino acid residues Pro (δ 4.60 ppm), Asp (δ 4.73 ppm) and Phe (δ 5.20 - 5.11); and (iii) 2 doublets integrating to 1 proton each at δ 5.16 and 5.07 ppm arising from the benzyl protecting group of Asp. The 13C NMR spectrum shows 5 carbons in the δ 176 - 170 ppm region, supporting the structure of 13 which has 1 ester and 4 amide linkages. HRMS (ES+) data showing m/z 535.2560 (MH+) confirms the formula of cyclic tetrapeptide 13, as opposed to dimeric or oligomeric structures which give the similar NMR spectra. The low yield of cyclic tetrapeptide 13 can be attributed to the formation of its dimer 14 as a more polar by-product (24%) due to intermolecular instead of intramolecular peptide coupling. Hence, the yield of the cyclic tetrapeptide is likely to be optimised with increased dilution, as lower concentration reduces the occurrence of the undesired intermolecular cyclisation. The 1H and 13C NMR spectra of dimer 14 are similar to 13, with slight variations in chemical shifts. Hence, the most compelling evidence for the structure of the dimer is the HRMS (ES+) indicating twice the mass of the tetrapeptide monomer at m/z 1069.5006 (MH+).

Hydrogenolysis of the benzyl ester protecting group in 13 with Pd-C catalyst in methanol afforded chlamydocin-acid analog 4 (49%), with its methyl ester 4a as a less polar by-product (40%). Due to mass and solubility constraints, NMR spectra were not obtained for chlamydocin-acid analog 4. HRMS (ES+) showing the correct mass m/z 445.2078 (MH+) was obtained before putting the acid through to the coupling reaction. The structure of its methyl ester 4a was elucidated by 1H NMR spectral evidence indicating (i) the loss of the benzyl protecting group, as marked by the decrease in number of aromatic protons and the disappearance of the peaks at δ 5.16 and 5.07 ppm, and (ii) the appearance of a signal at δ 3.69 ppm integrating to 3 protons. The 13C NMR spectrum showed a decrease in number of aromatic carbons, as well as replacement of the signal at δ 66.7 ppm (OCH2Ph) with another at δ 52.0 ppm (OCH3). HRMS data confirms the structure of 4a as shown in Fig 7.

3.3 Synthesis of Chlamydocin-Benzamide Amide Analog 1

As highlighted in Scheme 8, the final steps to chlamydocin-benzamide amide analog 1 involve the coupling of amine 3 with acid 4, followed by Boc deprotection.

Scheme 8 Synthesis of target compound 1. Reagents and conditions: a) HOBt (1.0 eq), DCC (1.2 eq), DIEA (1.0 eq), DMF, 0°C → rt, overnight; (b) TFA, 0°C, 2 h

Peptide coupling of amine 3 and acid 4 yielded the protected chlamydocin-benzamide amide analog 15 (22%). The low yield can be attributed to the formation of side products during coupling. Compound 16 was identified by comparing its spectral properties with amine 3 and methyl ester 4a. For the 1H NMR spectrum, the same peaks are observed with a slight variation in chemical shift and the loss of the methoxy proton peak at δ 3.69 ppm and also one N-H proton, indicating the presence of the desired product. Its 13C NMR spectrum showed 7 peaks in the δ 170 - 150 ppm region characteristic of amide carbons, fitting the structure of amide 15. The formula was confirmed by HRMS.

Finally, Boc deprotection with TFA to afford target compound 1 in 97% yield. In comparison with the 1H NMR spectrum of compound 15, target 1 lacks the strong peak at δ 1.49 ppm belonging to the methyl protons on the Boc protecting group. Also, its 13C NMR spectrum showed a loss of one amide carbon peak at δ 154.7 ppm, one tertiary carbon peak at δ 81.4 ppm and one methyl peak at δ 28.3 ppm as compared to compound 15. The formula was confirmed by HRMS.

3.4 Synthesis of Chlamydocin-Aldehyde Moiety

For the synthesis of chlamydocin-aldehyde analog 6, oxidative cleavage of chlamydocin-alkene analog 7 using osmium tetroxide and sodium periodate was employed. This method was preferred to ozonolysis due to precedent work by Bhuiyan et al.26 (Scheme 1) in oxidising terminal alkenes attached to the chlamydocin framework.

The oxidative cleavage was first attempted in a one-pot reaction as shown in Scheme 9. This procedure was chosen because of its ease and shorter duration as compared to a two-step route. However, the desired product aldehyde 6 was not obtained. Although the resulting product 6a (38%), has the same mass as aldehyde 6, the presence of the aldehyde was eliminated due to (i) the lack of a peak in the 9-10 ppm region of the 1H NMR spectrum, which is characteristic of aldehyde protons, (ii) the lack of a peak in the 190-200 ppm region of the 13C NMR spectrum, typical of aldehyde carbons, and (iii) the lack of a peak in the 1720-1750 cm-1 region of the IR spectrum, indicative of the aldehyde C=O stretch.

To investigate the source of this problem, the dihydroxylation and cleavage reactions were separated, allowing the faulty step to be pinpointed. Thus, the second attempt at the synthesis of aldehyde 6 followed the two-step procedure shown in Scheme 10. Dihydroxylation of alkene 7 with OsO4 and co-oxidant NMO gave diol 16 as a mixture of diastereomers in 72% yield. This confirms that dihydroxylation was successful. However, periodate cleavage of the diol disappointingly yielded product 6a (90%) again, instead of the desired aldehyde 6. Still, the overall yield over two steps is 65%, higher than that obtained from the one-pot procedure.

Product 6a was identified as a 75:25 mixture of γ-hydroxylactams formed by the reversible cyclisation of the aldehyde onto the amide (Fig 8). Precedent for the formation of γ-hydroxylactams via oxidative cleavage of pent-4-enoic acid amide motifs was reported by Baldwin, Freeman and Schofield37, in which a mixture of the aldehyde and both diastereomeric γ-hydroxylactam forms was obtained (Scheme 11).

The γ-hydroxylactam structure is supported by the NMR and IR spectra as observed from (i) the peaks at 5.64 and 5.56 ppm in the 1H NMR spectrum, belonging to the NCHOH proton of the major and minor diastereomers respectively, (ii) the quartet signals at 2.85 and 3.48 ppm, as well as doublets at 2.05 and 2.43 ppm, belonging to the NCH2CHOH protons, (iii) the peaks at 73.9 and 60.5 ppm in the 13C NMR spectrum indicative of sp3 carbons with a C-O bond, and (iv) a strong band at 1698 cm-1 in the IR spectrum, shifted to higher energy from the other amide C=O stretches at 1660 cm-1, indicative of a ring-constrained amide system.

3.5 Synthesis of Chlamydocin-Benzamide Amine Analog 2

Originally, the synthesis of target compound 2 was proposed via reductive amination of hypothetical aldehyde 6 with amine 3 to form amine 17, followed by Boc deprotection as shown in Scheme 12. Sodium triacetoxyborohydride is a mild reducing agent which should preferentially reduce the imine, formed by nucleophilic attack of the amine on the aldehyde, in the presence of aldehyde.

Scheme 12 Proposed synthesis of target compound 2. Reagents and conditions: (a) 1. amine 3 (1.1 eq), CH3COOH (1.5 eq), 2 h, 2. NaBH(OAc)3 (1.1 eq), 2 h; (b) TFA, 0°C

It was hypothesised that γ-hydroxylactam 6a could also undergo reductive amination by proceeding through its aldehyde form 6 as shown in the right of Scheme 13. An equilibrating mixture of γ-hydroxylactam 6a and aldehyde 6 should exist in solution, with aldehyde 6 reacting with amine 3 to form the imine in a reversible reaction. Reduction of the imine by sodium triacetoxyborohydride then forms amine 17, shifting the equilibrium position to the right and driving the reaction forward. A competing reaction pathway is unimolecular nucleophilic substitution (SN1) at the hydroxyl group of γ-hydroxylactam 6a, shown in the left of Scheme 11. Protonation of the hydroxyl group by acetic acid creates water, a good leaving group, which is lost to form an iminium ion. Amine 3 attacks the iminium to form substitution product 17a. As the C-N bond in the lactam is relatively unreactive towards cleavage, sodium triacetoxyborohydride is not expected to open the lactam ring. Thus, a stronger reducing agent is likely to be required to convert 17a into the desired amine 17.

Scheme 13 Hypothetical reaction mechanism for the proposed reductive amination of γ-hydroxylactam 6a

However, when reductive amination was attempted on γ-hydroxylactam 6a under the conditions described in Scheme 12, no reaction occurred, and the starting materials were recovered. This was attributed to the poor solubility of the compounds in 1,2-dichloroethane, leading to a low effective concentration of reactants in solution and thus inhibiting the reaction.

Scheme 14 Attempted reductive amination of γ-hydroxylactam 6a. Reagents and conditions: 1. amine 3 (1.5 eq), CH3COOH (1.5 eq), 2 h; 2. NaBH(OAc)3 (1.2 eq), 70°C, 1 h à rt, 3 days

Hence, the procedure was repeated under the reaction conditions shown in Scheme 14. DMF, a more polar solvent which dissolved all starting materials, was used. Despite the increased solubility of the reactants, the desired reaction was not observed, and starting materials were again recovered. However, a new compound benzamide 18 was also isolated. This is the product of a transamidation side reaction (Scheme 15) between benzamide 3 and DMF, with 89% conversion of benzamide 3.

Scheme 15 Proposed reaction mechanism for the transamidation side reaction between benzamide 3 and DMF under acidic conditions to generate benzamide 18

The same reaction was attempted at room temperature. Unfortunately, the desired reaction did not occur and starting material was recovered as before. However, the yield of benzamide 18 decreased, indicating that the transamidation side reaction is promoted by heating. The transamidation of DMF was previously studied by Musci, Chass and Csizmadia38 under basic conditions and with heating to 120°C, as shown in Scheme 16.

Scheme 16 Transamidation of DMF under basic conditions. Reagents and conditions: NaOMe, DMF, 120°C, 2 h [Musci et al.]

The absence of compound 17 suggests that γ-hydroxylactam 6a is highly stable with respect to aldehyde 6 and that the equilibrium constant between the two forms (shown in Fig 8) lies strongly towards the γ-hydroxylactam form. In addition, the absence of compound 17a also implies that the SN1 pathway is also not viable.

The reductive amination of γ-hydroxylactam 6a has thus far proved difficult, suggesting that alternative methods to target 2 should be employed instead. Unfortunately, due to time constraints these methods have not been attempted. However, they are outlined under Conclusions and Future Work as viable areas for further research.

4. Conclusions and Future Work

This report presents the successful preparation of a chlamydocin-benzamide amide target via a convergent approach. The compound will be screened for HDAC inhibitory activity against various HDAC enzymes by Mr. Rob Felstead to determine its potency and selectivity profile; preliminary tests are underway. Conditional on the HDAC inhibitory activity of the target, variation of the capping group and/or the metal-binding fragment can be conducted to confer greater potency and/or selectivity to the analogs.

The challenges in the synthesis of a chlamyocin-benzamide amine target are also described in this report. The main obstacle arises from cyclisation of the aldehyde functional group onto an amide nitrogen atom to form a γ-hydroxylactam intermediate. Alternative strategies to the target are outlined below.

From γ-hydroxylactam 6a, reduction to cleave the C-N bond forms alcohol 19, which is then converted to triflate 20. Lastly, nucleophilic substitution by amine 3 should lead to amine 17 as shown in Scheme 17.

Scheme 17 Retrosynthetic analysis of intermediate 17 leading to benzamide 3 and γ-hydroxylactam 6a.

Alternatively, the reactive amide group can be protected before oxidation so that aldehyde 6 will be unable to cyclise to γ-hydroxylactam 6a. The para-methoxybenzyl (PMB) protecting group39 can be used under these circumstances, as it protects both amines and amides and is stable under oxidative conditions. Protection is achieved by reaction of para-methoxybenzylbromide with the amine/amide in DMF at room temperature overnight. As this condition is amenable to the Ae5 amino acid and chlamydocin-alkene analog 7, PMB can either be introduced specifically to the Ae5 residue before it is coupled to form the tetrapeptide, or to all reactive amide groups on chlamydocin-alkene analog 7 after formation. Deprotection requires hydrogenation with PdCl2 in ethyl acetate with acetic acid catalyst, thus PMB can be removed after reductive amination to obtain compound 17.

5. Experimental

General experimental methods

All reactions were carried out under an argon atmosphere with dry solvents under anhydrous conditions, unless otherwise noted. Solvents and reagents were purchased commercially and used without further purification, unless otherwise stated. DMF was stirred over MgSO4 for 24 h, filtered and distilled under reduced pressure prior to use and stored over molecular sieves (4Å) under an argon atmosphere. DIEA was distilled from CaH2 prior to use and stored under an argon atmosphere. Yields refer to chromatographically and spectroscopically (1H NMR) homogeneous materials, unless otherwise stated. Flash column chromatography was performed on silica gel (Merck Kieselgel 60 F254 230-400 mesh) according to the method of W.C. Still40. TLC was performed on glass backed plates pre-coated with silica (0.2 mm, 60 F254) which were developed using standard visualizing agents: UV fluorescence (254 & 366 nm) and potassium permanganate (VII)/Δ. Melting points were determined on a Reichert hotplate microscope and are uncorrected. Optical rotations were recorded on a Perkin-Elmer 241 polarimeter at 589 nm (Na D-line) with a path length of 0.5 dm. Concentrations (c.) are quoted in g/100 mL and specific rotations, , are quoted in 10-1 degcm2g-1 at the specified temperature, T (°C). Infrared spectra were recorded as thin films or as solutions in CHCl3 using ATR on a Perkin-Elmer Spectrum 100 FT-IR spectrometer. Only selected absorbances (νmax) are reported. 1H NMR spectra were recorded at room temperature at 400 MHz on Bruker AV-400 or DRX-400 instruments. When mixtures of two rotamers are present in the NMR spectra these are quoted as Hmaj and Hmin, and the ratio stated. Mixtures of unassigned diastereomers are referred to as Ha and Hb, and the ratio stated. Chemical shifts (δH) are quoted in parts per million (ppm), referenced to the appropriate residual solvent peak e.g. CHCl3 δH 7.26. Coupling constants (J) are reported to the nearest 0.1 Hz. 13C NMR spectra were recorded at 100 MHz on Bruker AV-400 or DRX-400 instruments. Chemical shifts () are quoted in ppm referenced to the appropriate solvent peak e.g. CDCl3 δH 77.0. Low resolution mass spectra (m/z) were recorded on either VG Platform II (CI+ and ES+) or VG AutoSpec (CI+, ES+) spectrometers, with only molecular ions (M+), and major peaks from fragmentation of molecular ions being reported with intensities quoted as percentages of the base peak. High resolution mass spectra were recorded on the VG AutoSpec spectrometer.

4-[(9H-Fluoren-9-ylmethoxycarbonylamino)methyl]benzoic acid (8)34.

To a solution of 4-aminomethylbenzoic acid (1.00 g, 6.62 mmol, 1.0 eq) in dioxane-H2O (1:1) (50 mL) at 0°C was added Na2CO3 (2.10 g, 19.9 mmol, 3.0 eq). The resulting mixture was stirred at 0°C for 10 min, then allowed to warm to room temperature. Fmoc chloride (1.71 g, 6.62 mmol, 1.0 eq) was added and the reaction mixture was stirred overnight. Upon acidification to pH 5 with 0.1 M HCl, the product was precipitated out and the suspension was filtered. The residue was washed with H2O and hexane, and dried overnight in a vacuum desiccator to yield acid 8 as a white solid (2.13 g, 86%). δH (DMSO-d6, 400 MHz): 7.90 (2 H, d, J 7.5, Fmoc-ArH), 7.86 (1 H, t, J 6.1, NH), 7.81 (2 H, d, J 8.0, CHCCOOH), 7.72 (2 H, d, J 7.4, Fmoc-ArH), 7.43 (2 H, t, J 7.4, Fmoc-ArH), 7.34 (2 H, t, J 7.3, Fmoc-ArH), 7.12 (2 H, d, J 8.0, CHCCH2NH), 4.35 (2 H, d, J 6.9, Fmoc-CH2O), 4.24 (1 H, t, J 6.8, Fmoc-CHCH2O), 4.19 (2 H, d, J 6.1, CH2NH) [lit.34 δH (DMSO-d6, 300 MHz): 12.84 (1 H, br s), 7.30-7.90 (12 H, m), 4.38 (2 H, m), 4.24 (2 H, m)]; m/z (ES-) 418 ([M+HCO2]-, 100%), 372.1250 ([M-H]-, 85. C23H18NO4 requires 372.1236, Δ = 3.8 ppm).

(2-Aminophenyl)carbamic acid tert-butyl ester (9)35.

To a solution of benzene-1,2-diamine (1.00 g, 9.25 mmol, 1.0 eq) in dichloromethane (30 mL) at 0°C was added an aqueous solution of Na2CO3 (0.78 g, 5.55 mmol, 0.6 eq) and NaCl (0.54 g, 0.925 mmol, 0.1 eq) in distilled water (20 mL), and the mixture was stirred at 0°C for 30 min. Boc anhydride (2.018 g, 9.25 mmol, 1.0 eq) was dissolved in dichloromethane (20 mL) and the solution was slowly added to the reaction mixture. The mixture was allowed to stir at 0°C for an additional 10 min, warmed to room temperature, then refluxed overnight. The mixture was then cooled to room temperature and extracted with dichloromethane (3 x 30 mL). The combined organic layers were washed with sat. NaHCO3 solution (50 mL) and sat. NaCl solution (50 mL), then dried over anhydrous MgSO4. The mixture was filtered, and the solvent was removed in vacuo. The crude product was purified by flash chromatography on a silica gel column eluted with EtOAc/hexane (1:3) to yield amine 9 as an off-white solid (1.30 g, 67%). mp 101-104°C (CHCl3); Rf 0.39 (EtOAc/hexane, 1:2); δH (CDCl3, 400 MHz): 7.22 (1 H, s, NHCCH), 6.95 (1 H, td, J 7.6, 1.4, NH2CHCH), 6.74 (2 H, t, J 7.6, NH2CCH, NHCCHCH), 6.13 (1 H, s, NH), 3.82 (2 H, br s, NH2), 1.45 (9 H, s, CH3) [lit.35 δH (CDCl3, 400 MHz): 7.28 (1 H, d, J 9.2), 7.01 (1 H, m), 6.78 (2 H, m), 6.21 (1 H, br s), 3.73 (2 H, br s), 1.51 (9 H, s)]; δC (CDCl3, 100 MHz): 153.9 (s), 140.0 (s), 126.1 (d), 124.8 (d), 119.6 (d), 117.6 (d), 80.5 (s), 28.4 (3q); m/z (CI+) 209.1288 (MH+, 100%. C11H17N2O2 requires 209.1290, Δ = - 1.0 ppm).

[2-(4-[(9H-Fluoren-9-ylmethoxycarbonylamino)methyl]benzoylamino)phenyl]carbamic acid tert-butyl ester (10).

To a solution of acid 8 (1.87 g, 5.00 mmol, 1.0 eq) and amine 9 (1.04 g, 5.00 mmol, 1.0 eq) in DMF (20 mL) under Ar atmosphere at 0°C was added HOBt (0.77 g, 5.00 mmol, 1.0 eq), DCC (1.24 g, 6.00 mmol, 1.2 eq) and DIEA (0.83 mL, 5.00 mmol, 1.0 eq). The resulting mixture was stirred at 0°C allowed to warm to room temperature and stirred overnight. The reaction mixture was filtered to remove urea and DMF was removed in vacuo. The residue was dissolved in ethyl acetate (20 mL) and washed with 10% citric acid (20 mL), 4% NaHCO3 (20 mL) and sat. NaCl (20 mL), then dried over anhydrous MgSO4. The mixture was then filtered and solvent was removed from the filtrate in vacuo. The residue was purified by flash column chromatography on a silica gel column eluted with EtOAc/hexane (2:3) to yield amide 10 as an off-white solid (1.07 g, 38%). mp 88-91°C (CHCl3); Rf 0.41 (EtOAc/hexane, 1:1); νmax(CHCl3)/cm-1 3321 br (NH), 1703 (COOC), 1509 and 1478 (C=C-C); δH (CDCl3, 400 MHz): 9.45 (1 H, br s, NH), 7.85 (2 H, d, J 7.9, ArH), 7.76 (2 H, d, J 7.5, ArH), 7.69 - 7.64 (1 H, m, NH), 7.63 - 7.48 (3 H, m, ArH), 7.43 - 7.27 (5 H, m, ArH), 7.22 - 7.08 (4 H, m, ArH), 5.81 (1 H, t, J 5.9, NH), 4.45 (2 H, d, J 6.9, OCH2), 4.32 (2 H, d, J 5.8, NCH2), 4.19 (1 H, t, J 6.7, CH), 1.49 (9 H, s, CH3). δC (CDCl3, 100 MHz): 165.9 (s), 156.8 (s), 154.7 (s), 143.9 (s), 142.8 (s) , 141.4 (s), 133.0 (2s), 130.7 (s), 130.4 (2s), 127.9 (2d), 127.8 (2d), 127.3 (2d), 127.1 (2d), 126.1 (2d), 125.7 (d), 125.5 (d), 125.1 (d), 124.8 (d), 124.5 (d), 120.1 (d), 81.1 (t), 66.8 (s), 47.3 (t), 44.5 (d), 28.4 (3q); m/z (ES+) 586 (MNa+, 100%), 564.2502 (MH+, 18. C34H34N3O5 requires 564.2498, Δ = 0.7 ppm), 508 (21).

[2-(4-Aminomethylbenzoylamino)phenyl]carbamic acid tert-butyl ester (3)30.

To a solution of amide 8 (258 mg, 0.458 mmol, 1.0 eq) in dichloromethane under Ar atmosphere was added 1,8-diazabicyclo[5.4.0]undec-7-ene (116 μL, 0.778 mmol, 1.7 eq), and the resulting mixture was stirred. The progress of reaction was monitored by TLC. Upon completion the mixture was washed with 5% Na2CO3/10% NaCl and 30% NaCl solutions, then dried over anhydrous MgSO4. The resulting mixture was filtered and dichloromethane was removed from the filtrate in vacuo. The residue was purified by flash column chromatography on a silica gel column eluted with methanol/dichloromethane (1:4) to yield amide 11 as a colourless film (147 mg, 94%). Rf 0.11 (methanol/dichloromethane 1:4); νmax(CHCl3)/cm-1 3283 br (NH), 1693 and 1658 (CONH), 1509 and 1478 (C=C-C); δH (CDCl3, 400 MHz): 9.22 (1 H, br s, NH), 7.91 (2 H, d, J 8.1, C(O)CCH), 7.73 (1 H, d, J 7.4, NHCCH), 7.40 (2 H, d, J 8.1, CH2CCH), 7.28 - 7.23 (1 H, m, NHCCH), 7.19 (1 H, td, J 7.6, 1.6, NHCCHCH), 7.13 (1 H, td, J 7.6, 1.6, NHCCHCH), 7.07 (1 H, s, NH), 3.94 (2 H, s, CH2NH2), 2.07 (2 H, br s, NH2), 1.50 (9 H, s, CH3); δC (CDCl3, 100 MHz): 165.7 (s), 154.7 (s), 145.9 (s), 132.9 (s), 130.7 (d), 130.3 (d), 127.8 (2d), 127.4 (2d), 126.0 (d), 125.8 (d), 125.7 (d), 124.5 (d), 81.2 (s), 45.7 (t), 28.3 (3q); m/z (ES+) 364.1631 (MNa+, 71%. C19H22N3O3Na requires 364.1637, Δ = - 1.6 ppm), 342 (MH+, 16), 286 (53), 242 (73), 224 (100).

1-{2-[2-(3-Benzyloxycarbonyl-2-tert-butoxycarbonylaminopropionylamino)-2-methyl-propionylamino]-3-phenylpropionyl}pyrrolidine-2-carboxylic acid tert-butyl ester, Boc-L-Asp(OBn)-Aib-L-Phe-D-Pro-OtBu (11).

To a solution of H-Aib-L-Phe-D-Pro-OtBu (403 mg, 1.00 mmol, 1.0 eq) and Boc-Asp(OBn)-OH (323 mg, 1.00 mmol, 1.0 eq) in DMF (10 mL) under Ar atmosphere at 0°C was added HOBt (153 mg, 1.00 mmol, 1.0 eq), N,N'-dicyclohexylcarbodiimide (248 mg, 1.20 mmol, 1.2 eq) and DIEA (165 μL, 1.0 mmol, 1.0 eq). The resulting mixture was stirred at 0°C allowed to warm to room temperature and stirred overnight. The reaction mixture was filtered to remove urea and DMF was removed in vacuo. The residue was dissolved in EtOAc (20 mL) and washed with 10% citric acid (20 mL), 4% NaHCO3 (20 mL) and sat. NaCl (20 mL), then dried over anhydrous MgSO4. The mixture was then filtered and solvent was removed from the filtrate in vacuo. The residue was purified by flash column chromatography on a silica gel column eluted with methanol/dichloromethane (1:24) to yield tetrapeptide 11 (a 73:27 mixture of rotamers) as a white foam (563 mg, 79%). Rf 0.39 (methanol/dichloromethane, 1:19); +38.7 (c 1.60, CHCl3); νmax(CHCl3)/cm-1 3331 (NH), 1727 (COOC), 1677 and 1638 (CONH), 1498 (C=C-C); δH (CDCl3, 400 MHz): 7.34 - 7.27 (7 H, m, ArH), 7.22 (1 H, d, J 2.4, ArHmin), 7.21 - 7.18 (2 H, m, ArHmaj), 7.17 (4 H, d, J 6.9, ArHmaj), 7.13 (1 H, s, ArHmin), 7.02 (1 H, s, NHmaj), 7.00 (0.4 H, d, J 8.5, NHmin), 6.99 (0.4 H, s, NHmin), 6.90 (1 H, d, J 7.9, NHmaj), 5.69 (1 H, d, J 8.0, Asp-C(O)CH2CHmaj) , 5.48 (0.4 H, d, J 8.5, Asp-C(O)CH2CHmin), 5.14 (0.7 H, d, J 4.5, Asp(OBn)-OCHmin2Ph), 5.11 - 5.03 (2 H, m, Asp(OBn)-OCHmax2Ph), 4.91 (0.4 H, d, J 8.4, Phe-PhCH2CHmin), 4.86 (1 H, dd, J 15.2, 7.6, Phe-PhCH2CHmaj), 4.67 (0.4 H, td, J 9.7, 3.8, Pro-NCHmin), 4.52 - 4.40 (1 H, m, NHmaj), 4.34 (04 H, s, NHmin), 4.15 (1 H, dd, J 8.1, 3.4, Pro-NCHmaj), 3.57 (0.4 H, d, J 4.8, Pro-NCHmin2), 3.51 (1 H, dd, J 15.8, 8.7, Pro-NCHmaj2), 3.12 (0.4 H, dd, J 14.5, 3.8, Pro-NCHmin2), 3.03 (0.7 H, d, J 4.2, Asp-C(O)CHmin2), 2.98 (2 H, dd, J 11.3, 5.8, Asp-C(O)CHmaj2), 2.94 - 2.82 (1 H, m, Pro-NCHmaj2), 2.75 (1 H, dd, J 13.3, 6.3, Phe-PhCHmaj2), 2.72 - 2.66 (1 H, m, Phe-PhCHmaj2), 2.24 (0.4 H, dtd, J 11.2, 8.2, 8.0, Phe-PhCHmin2), 2.17 - 2.07 (0.4 H, m, Phe-PhCHmin2), 1.91 (0.4 H, d, J 3.4, Pro-CHmin2), 1.90 - 1.86 (1 H, m, Pro-CHmaj2), 1.86 - 1.82 (1 H, m, Pro-CHmaj2), 1.81 - 1.77 (1.4 H, m, Pro-CHmaj2, Pro-CHmin2), 1.77 - 1.75 (0.4 H, m, Pro-CHmin2), 1.70 - 1.60 (1 H, m, Pro-CHmaj2), 1.58 (0.4 H, d, J 4.1, Pro-CHmin2) 1.46 (6 H, s, Aib-CHmaj3, Boc-CHmin3), 1.43 (9 H, s, Boc-CHmaj3), 1.42 (6 H, s, Aib-CHmaj3, Boc-CHmin3), 1.38 (9 H, s, Boc-CHmaj3), 1.34 (0.9 H, s, Aib-CHmin3), 1.31 (0.8 H, s, Aib-CHmin3); δC (CDCl3, 100 MHz): 173.3, 172.0, 170.8, 170.0, 169.8, 169.5, 136.6, 135.4, 129.5, 129.3, 128.6, 128.3, 128.3, 126.8, 126.6, 81.0, 80.5, 66.9, 66.9, 60.0, 59.6, 57.0, 52.5, 51.9, 51.0, 46.7, 46.6, 39.0, 37.2, 36.1, 34.0, 31.2, 29.0, 28.3, 27.9, 25.6, 25.4, 25.0, 24.9, 24.7, 24.3, 22.4; m/z (ES+) 731 (MNa+, 100%), 709.3789 (MH+, 17. C38H53N4O9 requires 709.3813, Δ = - 3.4 ppm).

1-{1-[1-Benzyl-2-(2-carboxypyrrolidin-1-yl)-2-oxo-ethylcarbamoyl]-1-methylethylcarbamoyl}-2-benzyloxycarbonylethylammonium trifluoroacetate, H-L-Asp(OBn)-Aib-L-Phe-D-Pro-OH·TFA (12).

Tetrapeptide 11 (485 mg, 0.684 mmol, 1.0 eq) was dissolved in TFA (3.0 mL) at 0°C and kept for 2 h. After evaporation of TFA, the residue was solidified by trituration with diethyl ether. The resulting mixture was filtered, and the residue dissolved in chloroform. The solvent was removed in vacuo to yield tetrapeptide·TFA salt 12 (a 77:23 mixture of rotamers) as a colourless film (369 mg, 81%). mp 206-208°C (CHCl3); +38.6 (c 0.88, H2O); νmax(neat)/cm-1 1730 (COOC), 1665 and 1633 (CONH), 1528 and 1499 (C=C-C); δH (D2O, 400 MHz): 7.40 (14 H, s, ArHmaj), 7.38 (4 H, d, J 2.9, ArHmin), 7.29 (9 H, dd, J 14.8, 7.3, ArHmaj), 7.25 (4 H, s, ArHmin), 7.24 - 7.15 (7 H, m, ArHmaj), 5.21 (2 H, d, J 2.4, Asp(OBn)-OCHmin2Ph), 5.18 (6 H, s, Asp(OBn)-OCHmaj2Ph), 4.88 (3 H, d, J 7.0. Asp-C(O)CH2CHmaj),4.62 (1 H, dd, J 10.0, 4.2, Asp-C(O)CH2CHmin), 4.22 (3 H, t, J 6.2, Phe-PhCH2CHmaj), 4.18 (3 H, dd, J 9.2, 4.3, Pro-NCHmaj), 4.14 (1 H, t, J 6.4, Phe-PhCH2CHmin), 3.64 - 3.55 (3 H, m, Pro-NCHmaj2), 3.55 - 3.47 (1 H, m, Pro-NCHmin), 3.46 - 3.38 (1 H, m, Pro-NCHmin2), 3.11 (1 H, d, J 5.8, Pro-NCHmin2), 3.07 (3 H, d, J 5.8, Pro-NCHmaj2), 3.05 - 3.03 (2 H, m, Asp-C(O)CHmin2), 3.01 (6 H, d, J 7.2, Asp-C(O)CHmaj2), 2.96 (3 H, d, J 4.9, Phe-PhCHmaj2), 2.93 (3 H, dd, J 12.4, 6.4, Phe-PhCHmaj2), 2.88 (1 H, d, J 7.2, Phe-PhCHmin2), 2.81 (1 H, dd, J 14.3, 10.5, Phe-PhCHmin2), 2.21 - 2.11 (1 H, m, Pro-CHmin2), 2.10 - 1.97 (3 H, m, Pro-CHmaj2), 1.94 - 1.75 (9 H, m, Pro-CHmaj2), 1.72 - 1.59 (3 H, m, Pro-CHmin2), 1.31 (9 H, s, Aib-CHmaj3), 1.24 (9 H, s, Aib-CHmaj3), 1.20 (3 H, s, Aib-CHmin3), 1.11 (3 H, s, Aib-CHmin3); δC (D2O, 100 MHz): 176.0, 175.3, 170.8, 170.6, 167.2, 135.9, 135.0, 129.4, 129.3, 128.8, 128.7, 128.4, 127.3, 67.8, 59.6, 57.1, 52.7, 49.4, 47.5, 37.3, 34.9, 28.8, 24.0, 23.9, 23.8, 22.2; m/z (ES+) 553.2659 (M+, 100%. C29H37N4O7 requires 553.2662, Δ = - 0.5 ppm).

(2R, 5S, 11S)-5-Benzyl-8,8-dimethyl-4,7,10,13-tetraoxotetradecahydro-3a,6,9,12-tetraazacyclo-pentacyclododecen-11-yl)acetic acid benzyl ester, Cyclo(-L-Asp(OBn)-Aib-L-Phe-D-Pro-) (13).

To a DMF (50 mL) solvent, tetrapeptide·TFA salt 12 (285 mg, 0.428 mmol, 1.0 eq), HBTU (195 mg, 0.513 mmol, 1.2 eq) and DIEA (226 μl, 1.37 mmol, 3.2 eq) were added in 5 aliquots with 30 min time intervals while the solution was stirred vigourously. After the final addition, the reaction mixture was allowed to stir overnight. Upon completion, DMF was evaporated in vacuo. The residue was dissolved in EtOAc (20 mL) and washed with 10% citric acid (20 mL), 4% NaHCO3 (20 mL) and sat. NaCl (20 mL), then dried over anhydrous MgSO4. The mixture was then filtered and solvent was removed from the filtrate in vacuo. The residue was purified by flash column chromatography on a silica gel column eluted with methanol/dichloromethane (1:49) to yield:

Cyclic tetrapeptide 13 as a colourless film (107 mg, 47%). Rf 0.29 (methanol/dichloromethane, 1:24); -64.1 (c 1.65, CHCl3); νmax(CHCl3)/cm-1 3313 br (NH), 1732 (COOC), 1660 (CONH), 1630 and 1525 (C=C-C); δH (CDCl3, 400 MHz): 7.44 (1 H, d, J 10.2, NH), 7.38 - 7.27 (6 H, m, ArH), 7.25 (1 H, d, J 5.2, ArH), 7.23 - 7.17 (3 H, m, ArH), 6.08 (1 H, s, NH), 5.20 - 5.11 (1 H, m, Phe-PhCH2CH), 5.16 (1 H, d, J 12.2, Asp(OBn)-OCH2Ph), 5.07 (1 H, d, J 12.3, Asp(OBn)-OCH2Ph), 4.73 (1 H, ddd, J 10.1, 10.0, 4.9, Asp-C(O)CH2CH), 4.60 (1 H, dd, J 7.9, 1.4, Pro-NCH), 3.86 (1 H, ddd, J 9.5, 8.6, 4.9, Pro-NCH2), 3.28 - 3.15 (2 H, m, Pro-NCH2, Phe-PhCH2), 3.11 (1 H, dd, J 16.9, 10.0, Asp-C(O)CH2), 2.93 (1 H, dd, J 13.3, 5.9, Phe-PhCH2), 2.95 - 2.78 (1 H, m, NH), 2.65 (1 H, dd, J 16.9, 4.9, Asp-C(O)CH2), 2.35 - 2.22 (1 H, m, Pro-CH2), 2.22 - 2.07 (1 H, m, Pro-CH2), 2.02 (1 H, s, NH), 1.81 - 1.64 (2 H, m, Pro-CH2), 1.72 (3 H, s, Aib-CH3), 1.30 (3 H, s, Aib-CH3); δC (CDCl3, 100 MHz): 175.6 (s), 173.4 (s), 173.0 (s), 171.5 (s), 170.2 (s), 137.0 (s), 135.4 (s), 129.0 (2d), 128.6 (2d), 128.6 (2d), 128.4 (s), 128.3 (s), 126.7 (2d), 66.7 (t), 58.8 (d), 57.7 (d), 53.4 (d), 50.6 (s), 47.0 (t), 35.8 (t), 34.3 (t), 26.3 (q), 25.0 (q), 24.8 (t), 23.3 (t); m/z (ES+) 557 (MNa+, 100%), 535.2560 (MH+, 25. C29H35N4O6 requires 535.32557, Δ = 0.6 ppm).

Its dimer cyclo(-L-Asp(OBn)-Aib-L-Phe-D-Pro-L-Asp(OBn)-Aib-L-Phe-D-Pro) 14 as a pale brown solid (56 mg, 24%). mp 127-130°C (CHCl3); Rf 0.14 (methanol/dichloromethane, 1:24); +84.3 (c 1.11, CHCl3); νmax(CHCl3)/cm-1 3330 br (NH), 1736 (COOC), 1636 (CONH), 1530 and 1454 (C=C-C); δH (CDCl3, 400 MHz): 7.82 (1 H, d, J 7.1, NH), 7.50 (2 H, s, NH), 7.37 - 7.20 (20 H, m, ArH), 5.16 (2 H, d, J 12.3, Asp(OBn)-OCH2Ph), 5.11 (2 H, d, J 12.3, Asp(OBn)-OCH2Ph), 4.72 - 4.54 (2 H, m, Asp-C(O)CH2CH), 4.19 (2 H, dd, J 5.8, 1.3, Phe-PhCH2CH), 3.70 - 3.56 (2 H, m, Pro-NCH2), 3.14 - 2.96 (6 H, m, Pro-NCH2, Phe-PhCH2, Asp-C(O)CH2), 2.96 - 2.86 (2 H, m, Phe-PhCH2), 2.63 (2 H, dd, J 15.9, 7.6, Asp-C(O)CH2), 2.31 (1 H, br s, NH), 2.06 (2 H, br s, NH), 1.76 (2 H, ddd, J 15.1, 8.5, 8.5, Pro-CH2), 1.70 - 1.57 (2 H, m, Pro-CH2), 1.55 (6 H, s, Aib-CH3), 1.46 (6 H, s, Aib-CH3); δC (CDCl3, 100 MHz): 175.1, 171.9, 171.7, 171.3, 170.4, 136.0, 135.7, 129.3, 128.5, 128.2, 127.3, 66.6, 60.8, 56.9, 53.9, 50.9, 46.9, 38.1, 35.1, 27.7, 25.4, 24.5; m/z (ES+) 1091 (MNa+, 100%), 1069.5006 (MH+, 21. C58H69N8O12 requires 1069.5035, Δ = - 2.7 ppm).

(2R, 5S, 11S)-(5-Benzyl-8,8-dimethyl-4,7,10,13-tetraoxotetradecahydro-3a,6,9,12-tetraazacyclo-pentacyclododecen-11-yl)acetic acid, Cyclo(-L-Asp-Aib-L-Phe-D-Pro-) (4).

To a solution of cyclic tetrapeptide 13 (78 mg, 0.15 mmol, 1.0 eq) in methanol (5 mL) was added 10% Pd-C (20 mg). The mixture was hydrogenated at room temperature over 84 h. The reaction mixture was then filtered over Celite to remove Pd/C and methanol was removed from the filtrate in vacuo. The residue purified by flash column chromatography on a silica gel column eluted with methanol/dichloromethane (3:7) to obtain:

Acid 4 as a white powder (32 mg, 49%). Rf 0.28 (methanol/dichloromethane, 1:4); m/z (ES+) 467 (MNa+, 73%), 445.2078 (MH+, 100. C22H29N4O6 requires 445.2087, Δ = - 2.0 ppm).

Its methyl ester cyclo(-L-Asp(OMe)-Aib-L-Phe-D-Pro-) 4a (27 mg, 40%) as a white solid. mp 106-110°C (CHCl3); Rf 0.83 (methanol/dichloromethane, 1:4); -84.0 (c 1.00, CHCl3); νmax(CHCl3)/cm-1 3322 br (NH), 1738 (COOC), 1661 (CONH), 1525 and 1437 (C=C-C); δH (CDCl3, 400 MHz): 7.39 (1 H, d, J 10.3, NH), 7.32 (1 H, d, J 10.5, Phe-ArH), 7.30 - 7.27 (1 H, m, Phe-ArH), 7.26 - 7.16 (3 H, m, Phe-ArH), 6.01 (1 H, s, NH), 5.17 (1 H, td, J 10.1, 5.8, Phe-PhCH2CH), 4.74 - 4.67 (1 H, m, Asp-C(O)CH2CH), 4.67 - 4.60 (1 H, m, Pro-NCH), 3.92 - 3.81 (1 H, m, Pro-NCH2), 3.69 (3 H, d, J 3.4, Asp(OAc)-CH3), 3.29 - 3.18 (2 H, m, Pro-NCH2, Phe-PhCH2), 3.03 (1 H, dd, J 16.9, 9.6, Asp-C(O)CH2), 2.94 (1 H, dd, J 13.5, 5.7, Phe-PhCH2), 2.57 (1 H, dd, J 16.9, 5.1, Asp-C(O)CH2), 2.33 (1 H, tt, J 6.1, 5.1, Pro-CH2), 2.18 (1 H, dq, J 11.2, 8.1, Pro-CH2), 1.76 (3 H, d, J 5.5, Aib-CH3), 1.88 - 1.61 (2 H, m, Pro-CH2), 1.35 (3 H, s, Aib-CH3); δC (CDCl3, 100 MHz): 175.7 (s), 173.4 (s), 173.0 (s), 171.6 (s), 170.9 (s), 137.0 (s), 129.0 (2d), 128.6 (2d), 126.8 (s), 58.9 (d), 57.8 (d), 53.5 (d), 52.0 (q), 50.6 (s), 47.0 (t), 35.8 (t), 33.9 (t), 26.4 (q), 25.0 (q), 24.8 (t), 23.4 (t); m/z (ES+) 481 (MNa+, 100%), 459.2243 (MH+, 53. C23H31N4O6 requires 459.2244, Δ = - 0.2 ppm).

{2-[4-({2-[(2R, 5S, 11S)-5-Benzyl-8,8-dimethyl-4,7,10,13-tetraoxotetradecahydro-3a,6,9,12-tetraazacyclopentacyclododecen-11-yl]acetylamino}methyl)benzoylamino]phenyl}carbamic acid tert-butyl ester (15).

To a solution of acid 4 (32 mg, 0.072 mmol, 1.0 eq) and amine 3 (25 mg, 0.072 mmol, 1.0 eq) in DMF (20 mL) under Ar atmosphere at 0°C was added HOBt (11 mg, 0.072 mmol, 1.0 eq), DCC (18 mg, 0.086 mmol, 1.2 eq) and DIEA (12 μL, 0.072 mmol, 1.0 eq). The resulting mixture was stirred at 0°C allowed to warm to room temperature and stirred overnight. The reaction mixture was filtered to remove urea and DMF was removed in vacuo. The residue was dissolved in ethyl acetate (20 mL) and washed with 10% citric acid (20 mL), 4% sodium bicarbonate (20 mL) and sat. NaCl (20 mL), then dried over anhydrous MgSO4. The mixture was then filtered and solvent was removed from the filtrate in vacuo. The residue was purified by flash column chromatography on a silica gel column eluted with EtOAc/hexane (9:1) to yield protected chlamydocin-benzamide analog 15 as a colourless film (12 mg, 22%). Rf 0.73 (methanol/EtOAc/hexane, 3:4:3); -60.0 (c 0.83, CHCl3); νmax(CHCl3)/cm-1 3308 br (NH), 1662 (CONH), 1525 and 1449 (C=C-C); δH (CDCl3, 400 MHz): 9.27 (1 H, br s, Bam-NH), 7.77 (2 H, d, J 8.1, Bam-C(O)CCH), 7.72 (1 H, d, J 6.9, Bam-NHCCH), 7.53 (1 H, d, J 10.1, NH), 7.34 - 7.24 (4 H, m, ArH), 7.23 - 7.19 (5 H, m, ArH), 7.15 (1 H, dd, J 7.6, 1.8, Bam-NHCCH), 7.13 (1 H, s, NH), 6.69 (1 H, t, J 4.9, NH), 6.42 (1 H, s, NH), 5.16 (1 H, td, J 10.1, 5.5, Phe-PhCH2CH), 4.85 (1 H, td, J 9.4, 5.8, Asp-C(O)CH2CH), 4.63 - 4.54 (1 H, m, Pro-NCH), 4.48 - 4.33 (2 H, m, Bam-CH2NH), 3.93 - 3.80 (1 H, m, Pro-NCH2), 3.26 - 3.13 (2 H, m, Pro-NCH2, Phe-PhCH2), 2.91 (1 H, dd, J 13.4, 5.5, Phe-PhCH2), 2.78 (1 H, dd, J 14.8, 9.4, Asp-C(O)CH2), 2.59 (1 H, dd, J 14.8, 5.8, Asp-C(O)CH2), 2.35 - 2.24 (1 H, m, Pro-CH2), 2.23 - 2.09 (1 H, m, Pro-CH2), 1.76 (3 H, s, Aib-CH3), 1.75 - 1.66 (2 H, m, Pro-CH2), 1.49 (9 H, s, Boc-CH3), 1.34 (3 H, s, Aib-CH3); δC (CDCl3, 100 MHz): 175.5, 173.7, 172.9, 171.4, 169.7, 165.4, 154.7, 142.1, 137.0, 133.2, 130.8, 130.0, 129.0, 128.6, 127.7, 127.4, 126.8, 126.0, 125.9, 125.8, 124.6, 81.4, 58.9, 57.8, 53.5, 51.2, 47.1, 43.0, 38.6, 36.0, 28.3, 26.3, 24.9, 24.8, 23.4; m/z (ES+) 790 (MNa+, 100%), 768.3712 (MH+, 17. C41H54N3O12 requires 768.3721, Δ = - 1.2 ppm).

N-(2-Aminophenyl)-4-({2-[(2R, 5S, 11S)-5-benzyl-8,8-dimethyl-4,7,10,13-tetraoxotetradeca-hydro-3a,6,9,12-tetraazacyclopentacyclododecen-11-yl]acetylamino}methyl)benzamide (1).

Protected chlamydocin-benzamide analog 15 (12.3 mg, 0.0160 mmol, 1.0 eq) was dissolved in TFA (2.0 mL) at 0°C and kept for 2 h. After evaporation of TFA, the residue was solidified by trituration with ether. The resulting mixture was filtered, and the residue dissolved in chloroform. The solvent was removed in vacuo, and the residue was purified by flash column chromatography on a silica gel column eluted with methanol/dichloromethane (1:9) to yield the target chlamydocin-benzamide amide analog 1 as a white solid (10.5 mg, 97%). Rf 0.39 (methanol/dichloromethane, 1:9); -40.0 (c 0.70, CHCl3); νmax(CHCl3)/cm-1 3285 br (NH), 1679 and 1658 (CONH), 1527 and 1456 (C=C-C); δH (CDCl3, 500 MHz): 8.20 (1 H, s, NH), 7.73 (2 H, d, J 7.3, Bam-C(O)CCH), 7.49 (1 H, d, J 10.1, NH), 7.34 (1 H, d, J 10.4, NH), 7.30 (1 H, d, J 8.2, Bam-NH2CCH), 7.28 - 7.26 (1 H, m, ArH), 7.26 - 7.17 (5 H, m, ArH), 7.18 (1 H, s, NH), 7.08 (1 H, td, J 7.9, 0.9, Phe-ArH), 6.82 (2 H, d, J 7.9, Bam-NHCCHCH, Bam-NH2CCHCH), 6.79 (1 H, d, J 9.2, Bam-NHCCH), 6.42 (1 H, s, NH), 5.16 (1 H, td, J 10.1, 5.6, Phe-PhCH2CH), 4.84 (1 H, ddd, J 9.6, 9.5, 6.5, Asp-C(O)CH2CH), 4.57 (1 H, d, J 6.3, Pro-NCH), 4.41 (2 H, qd, J 15.6, 6.0, Bam-CH2NH), 3.86 (1 H, td, J 9.4, 4.9, Pro-NCH2), 3.27 - 3.12 (2 H, m, Pro-NCH2, Phe-PhCH2), 2.91 (1 H, dd, J 13.6, 5.6, Phe-PhCH2), 2.78 (1 H, dd, J 14.6, 9.1, Asp-C(O)CH2), 2.62 (1 H, dd, J 14.6, 5.9, Asp-C(O)CH2), 2.34 - 2.24 (1 H, m, Pro-CH2), 2.15 (1 H, ddd, J 15.3, 9.7, 5.9, Pro-CH2), 1.76 (3 H, s, Aib-CH3), 1.74 - 1.63 (2 H, m, Pro-CH2), 1.34 (3 H, s, Aib-CH3); δC (CDCl3, 100 MHz): 175.5, 173.8, 172.9, 171.5, 169.8, 165.7, 142.2, 140.7, 137.0, 133.2, 128.9, 128.6, 127.7, 127.4, 127.3, 126.8, 125.5, 124.9, 119.8, 118.4, 58.9, 57.8, 53.4, 51.3, 47.0, 42.9, 36.1, 35.8, 26.2, 24.9, 24.8, 23.4; m/z (ES+) 690 (MNa+, 76%), 668.3208 (MH+, 17. C36H42N7O6 requires 668.3197, Δ = 1.6 ppm).

(2R, 5S, 11S)-5-Benzyl-11-(2,3-dihydroxy-propyl)-8,8-dimethyl-decahydro-3a,6,9,12-tetraaza-cyclopentacyclododecene-4,7,10,13-tetraone (16).

To a solution of alkene 7 (54 mg, 0.13 mmol, 1.0 eq) in a mixture of Me2CO/MeCN/H2O (3:1:1, 5.0 mL) was added NMO (31 mg, 0.27 mmol, 2.1 eq) and 4% aq. OsO4 (100 μL,0.02 mmol, 0.2 eq). The bright yellow reaction mixture was stirred overnight. Upon completion, sat. Na2S2O3 (10 mL) was added whilst stirring, and the resulting mixture was extracted with EtOAc (3 x 15 mL). The combined organic layers were washed with sat. NaCl (30 mL) and dried over MgSO4. The mixture was then filtered and solvent was removed from the filtrate in vacuo. The residue was purified by flash column chromatography on a silica gel column eluted with methanol/dichloromethane (7:93) to yield diol 16 (an inseparable 53:47 mixture of diastereomers) as a white foam (43 mg, 73%). Rf 0.26 (methanol/dichloromethane, 1:9); νmax(CHCl3)/cm-1 3304 br (OH and NH), 1666 (CONH), 1527 and 1434 (C=C-C); δH (CDCl3, 400 MHz): 7.53 (1 H, t, J 11.2, NH), 7.40 (1 H, d, J 9.9, NH), 7.26 (2 H, t, J 7.2, Phe-ArH), 7.24 - 7.16 (3 H, m, Phe-ArH), 6.91 (0.43 H, s, OHa), 6.67 (0.48 H, s, OHb), 5.14 (1 H, td, J 10.0, 5.8, Phe-PhCH2CH), 4.76 - 4.65 (1 H, m, Pro-NCH), 4.64 (0.54 Hb, dd, J 10.1, 5.0, Ae5-NHCHCO), 4.58 (0.46 Ha, td, J 9.6, 5.1, NHCHCO), 3.85 (1 H, t, J 9.4, Pro-NCH2), 3.82 - 3.75 (0.51 H, m, Ae5-CHbOH), 3.71 - 3.67 (0.48 H, m, Ae5-CHaOH), 3.67 - 3.59 (1 H, m, CH2OH), 3.48 (1 H, dd, J 9.3, 6.5, CH2OH), 3.24 (1 H, dd, J 13.2, 10.3, Phe-PhCH2), 3.15 (1 H, dt, J 15.1, 7.9, Pro-NCH2), 2.93 (1 H, dd, J 13.4, 5.5, Phe-PhCH2), 2.37 - 2.20 (1 H, m, Pro-CH2), 2.20 - 2.02 (2 H, m, Pro-CH2, Ae5-CH2CHOH), 1.76 (3 H, s, Aib-CH3), 1.90 - 1.61 (4 H, m, Pro-CH2, Ae5-CH2CHOH, OH), 1.33 (3 H, s, Aib-CH3).; δC (CDCl3, 100 MHz): 175.4, 174.6, 174.4, 173.2, 173.1, 172.3, 172.0, 136.9, 129.0, 128.7, 126.8, 69.3, 68.7, 66.7, 66.6, 58.6, 58.6, 57.8, 53.3, 52.0, 46.8, 36.0, 33.1, 32.9, 26.0, 24.7, 23.7, 23.5; m/z (ES+) 483 (MNa+, 100%), 461.2399 (MH+, 13. C23H33N4O6 requires 461.2400, Δ = - 0.2 ppm).

(1S, 4R, 10S)-10-Benzyl-15-hydroxy-13,13-dimethyl-2,8,11,14-tetraazatricyclo[12.2.1.04,8] heptadecane-3,9,12,17-tetraone (6a).

Method A: To a solution of chlamydocin-alkene analog 7 (85.3 mg, 0.200 mmol, 1.0 eq) in THF/H2O (4:1, 7.5 mL) was added 4% aqueous solution of OsO4 (300 μL) over 5 in to produce a bronze solution which was stirred at room temperature for 10 min. The solution was then treated with NaIO4 (248 mg, 1.20 mmol, 5.8 eq), and the resulting mixture was stirred for 3 h. Upon completion, the reaction mixture was diluted with water (20 mL) and extracted with EtOAc (3 x 30 mL). The combined organic layers were washed with sodium thiosulphate (70 mL) and dried over MgSO4. The mixture was then filtered and solvent was removed from the filtrate in vacuo. The residue was purified by flash column chromatography on a silica gel column eluted with methanol/dichloromethane (1:19) to yield 6a (an inseparable mixture of aldehyde and lactam diastereomers) as a white solid (32.6 mg, 38%).

Method B: To a solution of diol 16 (41.4 mg, 0.0900 mmol, 1.0 eq) in THF/H2O (2:1, 9.0 mL) was added NaIO4 (57.8 mg, 0.270 mmol, 3.0 eq). The reaction was stirred for 1.5 h. Upon completion, the reaction mixture was stirred with aq. sat. Na2SO3 (20 mL) and extracted with diethyl ether (3 x 30 mL). The combined organic layers were washed with aq. sat. NaCl (70 mL) and dried over MgSO4. The mixture was then filtered and solvent was removed from the filtrate in vacuo. The residue was purified by flash column chromatography on a silica gel column eluted with ethanol/dichloromethane (3:97) to yield 6a (an inseparable 72:28 equilibrium mixture of lactam diastereomers) as a white solid (34.8 mg, 90%).

Rf 0.36 (methanol/dichloromethane, 1:9); νmax(CHCl3)/cm-1 3304 br (OH), 3227 br (NH), 1698 (lactam CONH), 1657 (CONH), 1544 (C=C-C); δH (CDCl3, 400 MHz): 8.12 (1 H, d, J 8.9, NHa), 6.96 (1 H, d, J 10.8, NHa), 5.92 (1 H, s, , NHa), 5.83 (0.4 H, d, J 8.6, NHb), 5.64 (1 H, dd, J 3.9, 2.0, CHaOH), 5.56 (0.4 H, dd, J 10.2, 6.7, CHbOH), 4.99 - 4.91 (1.4 H, m, CHaCH2CHOH) , 4.91 - 4.84 (0.4 H, m, CHbCH2CHOH), 4.80 (1 H, d, J 7.2, Phe- PhCH2CHa, Phe- PhCH2CHb), 4.75 (0.4 H, d, J 16.0, Pro-NCHb), 4.65 (1 H, dd, J 10.6, 8.4, Pro-NCHa), 4.08 (0.4 H, s, NHb), 3.61 (1 H, td, J 10.1, 2.8, Pro-NCHa 2) , 3.57 - 3.51 (0.4 H, m, Pro-NCHb 2), 3.48 (0.4 H, q, J 7.1, CHb2CHOH), 3.14 - 3.05 (1 H, m, Pro-NCHa 2), 3.04 (0.4 H, d, J 1.5, Pro-NCHb 2), 3.01 (1 H, d, J 5.6, Phe- PhCHa2), 2.97 (0.4 H, d, J 8.9, Phe- PhCHb2), 2.85 (1 H, q, J 8.8, CHa2CHOH), 2.62 (0.4 H, dd, J 15.2, 9.6, Pro-CHb 2), 2.55 - 2.44 (2 H, m, Pro-CHa 2), 2.43 (0.4 H, d, J 4.3, CHb2CHOH), 2.41 - 2.24 (0.8 H, m, Pro-CHb 2), 2.15 - 2.05 (1.4 H, m, Pro-CHa 2, Pro-CHb 2), 2.05 (1 H, d, J 14.4, CHa2CHOH) , 1.75 (1 H, dd, J 18.3, 9.8, Pro-CHa 2), 1.67 (5 H, s, Aib-CHa3, OHa, OHb), 1.58 (3H, s, Aib-CHa3), 1.56 (1.2 H, s, Aib-CHb3), 1.51 (1.4 H, s, Aib-CHb3); δC (CDCl3, 100 MHz): 175.6, 174.8, 174.6, 174.3, 173.6, 172.9, 136.5, 129.1, 128.9, 128.8, 128.7, 127.1, 127.1, 123.1, 81.2, 80.1, 73.9, 60.5, 59.8, 59.0, 58.7, 55.9, 54.1, 51.5, 51.3, 46.6, 46.5, 39.0, 38.2, 36.3, 34.2, 24.5, 23.9, 23.6, 23.5, 23.4, 23.1, 23.0; m/z (ES+) 451 (MNa+, 100%), 429.2147 (MH+, 10. C22H29N4O5 requires 429.2138, Δ = 2.1 ppm).

[2-(4-Formylaminomethyl-benzoylamino)-phenyl]-carbamic acid tert-butyl ester (18).

Rf 0.36 (methanol/dichloromethane, 1:9); νmax(CHCl3)/cm-1 3275 br (NH), 1662 (COOC), 1601 (CONH), 1510 and 1451 (C=C-C); δH (CDCl3, 400 MHz): 9.33 (1 H, s, CHO), 8.22 (1 H, s, NH), 7.85 (2 H, d, J 8.0, C(O)CCH), 7.74 (1 H, d, J 7.7, NHCCH), 7.30 (3 H, d, J 8.3, CH2CCH, NHCCH), 7.19 (2 H, qd, J 7.4, 3.8, NHCCHCH), 7.10 (1 H, s, NH), 6.49 (1 H, s, NH), 4.49 (2 H, d, J 6.0, CH2NH2), 1.51 (9 H, s, CH3); δC (CDCl3, 100 MHz): 165.4, 161.3, 154.7, 141.8, 133.3, 130.7, 130.1, 127.8, 127.7, 127.0, 126.1, 125.9, 125.7, 124.6, 81.4, 41.6, 28.3; m/z (ES+) 392.1592 (MNa+, 100%. C20H23N3O4Na requires 392.1586, Δ = 0.6 ppm).

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