WO2025114775A1 - Macrocyclic tubulin polymerization inhibitors as anticancer agents - Google Patents

Abstract

The present invention relates to the field of chemistry and biochemistry and in particular to the novel anticancer compounds, more particularly to antiproliferative agents and microtubule polymerization inhibitors. Even more particularly, the invention relates to novel analogs of natural antimitotic agent diazonamide A and to salts, pharmaceutical compositions, conjugates and a process of manifacture thereof and the potential use of the novel compounds for treatment of melanoma. A new series of compounds with high activity against cancer cells has been developed. The most active compounds from the series show cytotoxicity against several cancer cell lines with IC₅₀ = 12–90 nM.

Description

Macrocyclic tubulin polymerization inhibitors as anticancer agents

FIELD OF THE INVENTION

The present invention relates to the field of chemistry and biochemistry and in particular to the novel anticancer compounds, more particularly to antiproliferative agents and microtubule polymerization inhibitors. Even more particularly, the invention relates to novel analogs of natural antimitotic agent diazonamide A and to salts, pharmaceutical compositions, conjugates and a process of manifacture thereof and the potential use of the novel compounds for treatment of melanoma.

BACKGROUND OF THE INVENTION

Cancer is a major public health problem worldwide with more than 19 million new cancer cases diagnosed and 10 million lethal outcomes in 2020 [1], Growing incidence and high mortality together with increasing multidrug resistance towards cancer therapeutics [2] puts the development of new effective anticancer treatment among top priorities worldwide. Chemotherapeutics are the most effective means for the treatment of tumors. However, a majority of cancer chemotherapeutic agents currently used in clinics develops resistance overtime. Therefore, there still is high medical need for effective anticancer chemotherapeutic medicines.

The marine metabolite diazonamide A exerts nanomolar cytotoxicity against a range of human tumor cell lines. It has been demonstrated that diazonamide A prevents formation of the mitotic spindle during cell division by inhibition of microtubules assembly and, consequently, tubulin polymerization [3], However, high structural complexity of diazonamide A precludes its use in cancer therapy.

,

Decrease of structural complexity of diazonamide A without affecting its anticancer activity profile is possible as was demonstrated by the development of anticancer agent DZ-2384 [4, 5], A notable feature of DZ-2384 as a cancer chemotherapeutic agent is that in animal models it does not cause systemic toxicity typical for other antimitotic such as taxanes (paclitaxel), and vinca alkaloids (vinorelbine) [5], Although being structurally less complex as compared to diazonamide A, DZ-2384 still contains remarkable structural complexity with the chiral tetracyclic subunit EFGH imposing most of the synthetic challenges. Thus, the highly optimized synthesis of DZ-2384 required 13 synthetic steps with the key transformation being electrochemical macrocyclization to form the tetracycle EFGH that proceeded with poor 35% yield (48% based on recovered starting material) and afforded 2.7: 1 mixture of diastereomers that required separation by silica gel columnm chromatography [6],

The development of less-complex analogs of diazonamide A that lacks chiral tetracyclic subunit EFGH is highly desirable. Among numerous analogs of diazonamide A reported in the literature, there is a handful of macrocycles that lack the tetracyclic subunit EFGH (compounds A,B) [4], One example of diazonamide analog (compound D) with a tricyclic moiety EFG instead of the tetracycle EFGH has been also disclosed [4],

Recently we found that the replacement of the difficult-to-synthesize tetracycle subunit EFGH by aliphatic chain leads to the loss of activity as evidenced by remarkable reduction of tubulin binding afinity for the resulting macrocycles C as compared to DZ-2384 [7], On the other hand, we have recently demonstrated that the incorporation of oxindole subunit instead of the tetracycle subunit EFGH affords macrocycles E that are potent tubulin polymerization inhibitors with nanomolar citotoxicity against a series of cancer cell lines [8,9], Unfortunately, oxindole moiety-containing macrocycles B feature poor metabolic stability that hampers their application as anti-cancer agents.

SUMMARY OF THE INVENTION

In a first aspect, the invention features a method for the treatment of tumours, comprising administering to the human in need thereof a therapeutically effective amount of a compound or prodrug thereof, or pharmaceutically acceptable salt, hydrate, solvate, or polymorph of said compound or prodrug, wherein the compound is an inhibitor of microtubules assembly and tubulin polymerization.

In another aspect, the invention features a pharmaceutical composition for the treatment of tumors, comprising a therapeutically effective amount of a composition comprising (i) a compound or prodrug thereof, or pharmaceutically acceptable salt, hydrate, solvate, or polymorph of said compound or prodrug; and (ii) a pharmaceutically acceptable carrier, wherein the compound is an inhibitor of microtubules assembly and tubulin polymerization In another aspect, the invention features the use of a compound or prodrug thereof, or pharmaceutically acceptable salt, hydrate, solvate, or polymorph of said compound or prodrug, wherein the compound is an inhibitor of microtubules assembly and tubulin polymerization, in the manufacture of medicament for the treatment of tumors.

In another aspect, the invention features a compound or prodrug thereof, or pharmaceutically acceptable salt, hydrate, solvate, or polymorph of said compound or prodrug for the use in the treatment of tumors, wherein the compound is an inhibitor of microtubules assembly and tubulin polymerization.

In one embodiment the inhibitor of microtubules assembly and tubulin polymerization is a compound of general formula (I) or a pharmaceutically acceptable salt or conjugate thereof.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless otherwise stated, the following terms used in the specification and claims have the following meanings set out below.

It is to be appreciated that references to “treating” or “treatment” include prophylaxis as well as the alleviation of established symptoms of a condition. “Treating” or “treatment” of a state, disorder or condition therefore includes: (1) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a human that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition, (2) inhibiting the state, disorder or condition, Le., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or subclinical symptom thereof, or (3) relieving or attenuating the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms.

The term “alkyl” as used herein refers to a straight or branched alkyl chain having one to six (inclusive) carbon atoms. For example, the alkyl group may be part cyclic (cyclopropylmethyl), linear or branched (ethyl, //-propyl, iso-propyl, //-butyl, .scc-butyl, Zc/7-butyl).

The terms “alkylene,” “alkenylene,” or “alkynylene” as used herein refers to an alkyl, alkenyl, or alkynyl group that is positioned between and serves to connect two other chemical groups. For example, the term “Ci-ealkylene” means a linear saturated divalent hydrocarbon radical of one to six carbon atoms or a branched saturated divalent hydrocarbon radical of three to six carbon atoms, such as methylene, ethylene, propylene, 2-methylpropylene, pentylene, and the like. The term “C2- ealkenylene” means a linear divalent hydrocarbon radical of two to six carbon atoms or a branched divalent hydrocarbon radical of three to six carbon atoms, containing at least one double bond, for example, as in ethenylene, 2,4-pentadienylene, and the like. The term “C2-ealkynylene” means a linear divalent hydrocarbon radical of two to six carbon atoms or a branched divalent hydrocarbon radical of three to six carbon atoms, containing at least one triple bond, for example, as in ethynylene, propynylene, and butynylene and the like.

The term “cycloalkyl” as used herein refers to nonaromatic, saturated or partially unsaturated cyclic, bicyclic, tricyclic or polycyclic hydrocarbon groups having three to 12 carbon atoms. Cycloalkyl groups can contain fused rings. Fused rings are rings that share one or more common carbon atoms. Any ring atom can be substituted (e.g., with one or more substituents). Examples of G-xcycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, methylcyclohexyl, adamantyl, norbornyl and norbornenyl.

The term “alkenyl” refers to a straight or branched alkyl chain having two to six (inclusive) carbon atoms and one or more double bonds. Examples of alkenyl groups include, but are not limited to, allyl, propenyl, 2-butenyl and 3-hexenyl groups. One of the double bond carbons may optionally be the point of attachment of the alkenyl substituent.

The term “alkynyl” refers to a straight or branched hydrocarbon chain having two to six (inclusive) carbon atoms and one or more triple bonds. Examples of alkynyl groups include, but are not limited to, ethynyl, propargyl, and 3 -hexynyl. One of the triple bond carbons may optionally be the point of attachment of the alkynyl substituent.

The term “aryl” means a cyclic or polycyclic aromatic ring having from 5 to 12 carbon atoms. The term aryl includes both monovalent species and divalent species. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl and the like. In particular embodiment, an aryl is phenyl.

The term “arylCi-ealkyl” means an aryl group covalently attached to a Ci-ealkylene group, both of which are defined herein. Examples of arylCi-ealkyl groups include benzyl, phenylethyl, and the like.

The term “heteroaryl” means an aromatic mono-, or bi-cyclic ring incorporating one or more (for example 1-4, particularly 1, 2 or 3) heteroatoms selected from nitrogen, oxygen or sulfur. The term heteroaryl includes both monovalent species and divalent species. Examples of heteroaryl groups are monocyclic and bicyclic groups containing from five to twelve ring members, and more usually from five to ten ring members. The heteroaryl group can be, for example, a 5- or 6-membered monocyclic ring or a 9- or 10-membered bicyclic ring, for example a bicyclic structure formed from fused five and six membered rings or two fused six membered rings. Each ring may contain up to about four heteroatoms typically selected from nitrogen, sulfur and oxygen. Typically the heteroaryl ring will contain up to 3 heteroatoms, more usually up to 2, for example a single heteroatom. In one embodiment, the heteroaryl ring contains at least one ring nitrogen atom. The nitrogen atoms in the heteroaryl rings can be basic, as in the case of an imidazole or pyridine, or essentially non-basic as in the case of an indole or pyrrole nitrogen. In general the number of basic nitrogen atoms present in the heteroaryl group, including any amino group substituents of the ring, will be less than five.

Examples of heteroaryl include furyl, pyrrolyl, thienyl, oxazolyl, isoxazolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,3, 5 -triazenyl, benzofiiranyl, indolyl, isoindolyl, benzothienyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, benzothiazolyl, indazolyl, purinyl, benzofiirazanyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl, cinnolinyl, pteridinyl, naphthyridinyl, carbazolyl, phenazinyl, benzisoquinolinyl, pyridopyrazinyl, thieno[2,3-b]furanyl, 2H-furo[3,2-b]-pyranyl, 5H- pyrido [2, 3 -d] -o-oxazinyl, 1 H-pyrazolo [4,3 -d]-oxazolyl, 4H-imidazo[4, 5 -d]thiazolyl, pyrazino [2,3 - d]pyridazinyl, imidazo[2, l-b]thiazolyl, imidazo[l,2-b][l,2,4]triazinyl. “Heteroaryl” also covers partially aromatic bi- or polycyclic ring systems wherein at least one ring is an aromatic ring and one or more of the other ring(s) is a non-aromatic, saturated or partially saturated ring, provided at least one ring contains one or more heteroatoms selected from nitrogen, oxygen or sulfur. Examples of partially aromatic heteroaryl groups include for example, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 2-oxo-l,2,3,4-tetrahydroquinolinyl, dihydrobenzthienyl, dihydrobenzfuranyl, 2,3-dihydrobenzo[ l,4]dioxinyl, benzo[l,3]dioxolyl, 2,2-dioxo-l,3-dihydro-2-benzothienyl, 4, 5,6,7- tetrahydrobenzofuranyl, indolinyl, l,2,3,4-tetrahydro-l,8-naphthpyridinyl, 1,2, 3, 4- tetrahydropyrido[2,3-b]pyrazinyl and 3,4-dihydro-2H-pyrido[3,2-b][l,4]oxazinyl.

The term “heteroarylCi-ealkyl” means a heteroaryl group covalently attached to a Ci-ealkylene group, both of which are defined herein. Examples of heteroarylalkyl groups include pyridin-2- ylmethyl, 3-(benzofiiran-2-yl)propyl, and the like.

Where optional substituents are chosen from “one or more” groups it is to be understood that this definition includes all substituents being chosen from one of the specified groups or the substituents being chosen from two or more of the specified groups.

The phrase “compound of the invention” means those compounds which are disclosed herein, both generically and specifically. Compounds of the invention

In one aspect, the present invention relates to a compound of general formula (I) or a pharmaceutically acceptable salt or solvate thereof, as shown below:

general formula (I) wherein

R¹ represents optional substituent at quaternary carbon;

R² represents optional substituent at oxazole;

R³ represents optional substituent at aromatic subunit;

Q represents C1-2 alkylene group, C1-2 heteroalkylene group;

Compound with general formula (I), wherein:

R¹ is -CN, -C(=O)R⁴, -C(=O)OR⁴, -C(=O)N(R⁴)R⁵, -CH₂N(R⁴)R⁵, wherein:

R⁴, R⁵, independently represent, on each occasion when used herein, H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, arylCi-ealkyl, heteroaryl, heteroarylCi-ealkyl,

R⁴ and R⁵ taken together represent -V^A-W^A-X^A-Y^A-Z^A-, or -V^A-W^A-X^A-Y^A-, -V^A-W^A-X^A-, -

_V ^A-W^A- wherein:

V^A represents, oxygen, sulfur, -NR⁶, -CR⁶R⁷

W^A represents oxygen, sulfur, -NR⁶, -CR⁶R⁷

X^A represents oxygen, sulfur, -NR⁶, -CR⁶R⁷

Y^A represents oxygen, sulfur, -NR⁶, -CR⁶R⁷ Z^A represents oxygen, sulfur, -NR⁶, -CR⁶R⁷, wherein:

R⁶, R⁷, independently represent, on each occasion when used herein, H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, arylCi-ealkyl, heteroaryl, heteroarylCi-ealkyl,

R² is -F, -Cl, -Br, -I, -L-CF3, -L-CHF₂, -L-CFFF. -L-F, -L-OR⁸, -L-NR⁸R⁹, -L-C(=O)R⁸, -L- C(=N-OR⁸)R⁹, -L-C(=O)OR⁸, -L-C(=O)OCR⁸R⁹OC(=O)-L-R¹⁰, -L-C(=O)NR⁸R⁹, -L- C(=O)NR⁸-OR⁹, -L-CN, -L-NO2, -L-aryl, -L-arylCi-ealkyl, -L-heteroaryl wherein:

(a) Heteroaryl is selected from:

wherein:

R^H represent H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, arylCi-ealkyl, heteroaryl, heteroarylCi- ealkyl,

(b) R⁸, R⁹and R¹⁰ independently represent, on each occasion when used herein, H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, arylCi-ealkyl, heteroaryl, heteroarylCi-ealkyl

R⁸ and R⁹ taken together represent -V^B-W^B-X^B-Y^B-Z^B-, or -V^B-W^B-X^B-Y^B-, -V^B-W^B-X^B-, - V^B-W^B- wherein:

V^B represents, oxygen, sulfur, -NR¹¹, -CRⁿR¹²

W^B represents oxygen, sulfur, -NR¹¹, -CRⁿR¹²

X^B represents oxygen, sulfur, -NR¹¹, -CRⁿR¹²

Y^B represents oxygen, sulfur, -NR¹¹, -CRⁿR¹²

Z^B represents oxygen, sulfur, -NR¹¹, -CRⁿR¹² wherein:

R¹¹, R¹², independently represent, on each occasion when used herein, H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, arylCi-ealkyl, heteroaryl, heteroarylCi-ealkyl L represents -W^c-X^c-Y^c- or -W^c-X^c- or -W^c- wherein:

W^c is absent or represents oxygen, sulfur, -NR¹³, =CR¹³, -CR¹³R¹⁴

X^c represents oxygen, sulfur, -NR¹³, =CR¹³, -CR¹³R¹⁴

Y^c represents oxygen, sulfur, -NR¹³, =CR¹³, -CR¹³R¹⁴ wherein:

R¹³, R¹⁴ independently represent, on each occasion when used herein, H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, arylCi-ealkyl, heteroaryl, heteroarylCi-ealkyl

R¹³ and R¹⁴ taken together represent -V^D-W^D-X^D-Y^D-Z^D-, or -V^D-W^D-X^D-Y^D-, -V^D-W^D-X^D-, - V^D-W^D- wherein:

V^D represents, oxygen, sulfur, -NR¹⁵, -CR¹⁵R¹⁶

W^D represents oxygen, sulfur, -NR¹⁵, -CR¹⁵R¹⁶

X^D represents oxygen, sulfur, -NR¹⁵, -CR¹⁵R¹⁶

Y^D represents oxygen, sulfur, -NR¹⁵, -CR¹⁵R¹⁶

Z^D represents oxygen, sulfur, -NR¹⁵, -CR¹⁵R¹⁶ wherein:

R¹⁵, R¹⁶ independently represent, on each occasion when used herein, H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, arylCi-ealkyl, heteroaryl, heteroarylCi-ealkyl

Q represents -W^E-X^E-, or -W^E- wherein:

W^E represents oxygen, sulfur, -S(=O)-, -SO2-, -CH2-

X^E represents oxygen, sulfur, -S(=O)-, -SO2-, -CH2-

Compounds of formula (I) and disclosed embodiments thereof possess four or more asymmetric centers and therefore exist in different enantiomeric and diastereomeric forms. All optical isomers and stereoisomers of the compounds described herein, and mixtures thereof, are considered to be within the scope of the invention, including racemate, one or more enantiomeric forms, one or more diastereomeric forms, or mixtures thereof.

In one preferred embodiment, compound with general formula (I) possesses all four stereogenic centers with (5) absolute configuration.

In another aspect, the invention features compounds with general formula (I) obtainable by a method of synthesis as described herein, or a method of synthesis as described herein.

In another aspect, the invention features novel intermediates for the compound with general formula (I), as described herein, which are suitable for use in the methods of synthesis described herein.

In another aspect, the invention features the use of such novel intermediates, for the compound with general formula (I) according to the claim 1 as described herein, in the methods of synthesis described herein.

As will be appreciated by one of skill in the art, features and preferred embodiments of one aspect of the invention will also pertain to other aspects of the invention.

Description of the Invention

Natural marine metabolite diazonamide A is a highly potent cancer chemotherapy agent, which exerts its anti-tumor activity by preventing the formation of the mitotic spindle during cell division by inhibition of microtubules assembly and, consequently, tubulin polymerization [3], A structurally simplified diazonamide analog DZ-2384 is highly effective chemotherapy agent in the treatment of triple-negative breast cancer [10],

It has been surprisingly found that the tetracyclic subunit EFGH in the diazonamide A could be replaced by indane subunit to provide a novel class of macrocyclic diazonamide analogs of general formula (I) that displays nanomolar to low micromolar cytotoxicity against a wide panel of human tumor cell lines, including A2058 (metastatic melanoma), MDA-MB-435 (metastatic melanoma), U937 (myeloid leukemia) and MDA-MB-231 (breast adenocarcinoma). At the same time, compounds of General formula (I) are not cytotoxic towards normal human cell lines (such as human embryonic kidney cells (HEK-293). Furthermore, some of the compounds of general formula (I) show high plasma stability (see Table 4).

Results from tubulin polymerization experiments demonstrate that the compounds of general formula (I) bind to tubulin and disrupts tubulin polymerization dinamics in the same manner as other marketed tubulin polymerization inhibitors (vinblastine, vinorelbine). Flow cytometry analysis shows that compounds of general formula (I) induce strong cell cycle arrest in the G2/M phase, possessing identical effect to known antimitotic drugs such as vinorelbine (see Figure 2).

Compounds of general formula (I) are useful as cancer chemotherapy agents in the treatment of tumors including melanoma and metastatic melanoma. These macrocycles can be used for manufacturing of various pharmaceutical compositions, wherein they are present together with one or more pharmaceutically acceptable diluents, carriers or excipients.

Stereochemistry

Compounds of general formula (I) and disclosed embodiments thereof possess four or more asymmetric centers and therefore exist in different enantiomeric and diastereomeric forms. All optical isomers and stereoisomers of the compounds described herein, and mixtures thereof, are considered to be within the scope of the invention, including racemate, one or more enantiomeric forms, one or more diastereomeric forms, or mixtures thereof.

Examples of specific embodiments

The following examples further illustrate the invention, but should not be construed to limit the scope of the invention in any way.

The following compounds were prepared as an examples of the current invention:

Table 1

General Synthesis

The synthesis of diazonamide A analog DZA-129 is shown in Scheme 1.

Accordingly, nitrile 1 was reacted with freshly prepared organozinc species 2 in Pd-catalyzed Negishi cross-coupling with quantitative yield. Ester hydrolysis in 3 was followed by amide coupling with corresponding amine 4 giving amide 5. The key macrocyclization step in presence of K3PO4 furnished macrocycle 6 with diastereomeric ratio 98:2. After A-Boc cleavage with TFAthe resulting amine was coupled with fS')-2-hydroxy-3 -methylbutanoic acid fS'-H/VA; 7) to give analog DZA-129 (Scheme 1).

Scheme 1. Synthesis of analog DZA-129

Methyl (2 )-2-{[(tert-butoxy)carbonyl]amino}-3-(3-cyano-2,3-dihydro-lH-inden-5- yl)propanoate (3). Aryl bromide 1 (1.50 g; 6.75 mmol), Pd₂(dba)a (93 mg; 0.10 mmol; 0.02 equiv.) and SPhos (111 mg; 0.27 mmol; 0.04 equiv.) were dissolved in anhydrous DMF (5 mL) under argon atmosphere and freshly prepared solution of alkyl zinc iodide 2 (0.5 M in DMF; 20.3 mL; 10.13 mmol; 1.5 equiv.) (prepared as described in [11]) was added. The dark red solution was stirred at 65 °C for 3 h. Then the solution was cooled to room temperature and aqueous saturated NH4CI and EtOAc were added. Layers were separated and EtOAc was washed with IN HC1 aqueous solution and brine, dried (Na₂SO₄) and evaporated to dryness. The residue was purified by column chromatography on silica (10% to 50% EtOAc in hexanes) to afford 2.30 g (99%; dr ~1:1) of DZA- 129 as a yellow amorphous solid.

’H NMR (400 MHz, CDCh) 8 7.22 - 7.11 (m, 2H), 7.08 - 7.00 (m, 1H), 5.00 (d, J = 8.3 Hz, 1H), 4.63 - 4.51 (m, 1H), 4.11 - 4.03 (m, 1H), 3.75 (s, 1.5H), 3.73 (s, 1.5H), 3.21 - 2.98 (m, 3H), 2.98 - 2.83 (m, 1H), 2.65 - 2.52 (m, 1H), 2.42 - 2.29 (m, 1H), 1.42 (s, 4.5H), 1.43 (s, 4.5H). ¹³C NMR (101 MHz, CDCh) 6 172.4, 172.2, 155.2, 155.1, 141.84, 141.81, 138.2, 138.1, 135.6, 129.84, 129.79, 125.3, 125.2, 125.12, 121.13, 121.08, 80.2, 80.1, 54.7, 54.6, 52.53, 52.45, 38.23, 38.16, 34.54, 31.45, 31.4, 31.3, 28.42, 28.37. HRMS (m/z): Ci9H₂4N₂O₄Na [M+Na]⁺ found: 367.1644. Calculated: 367.1634.

General procedure A (amide formation):

Methyl ester was dissolved in THF. Separately solid LiOH*H₂O was dissolved in water and added to the reaction mixture. Emulsion was stirred at room temperature for 30 min, then aqueous HC1 (1 N) was added. The resulting mixture was extracted with EtOAc (x3), combined organic layers were washed with brine, dried (Na₂SO₄) and evaporated. To the resulting crude acid was added the corresponding amine (prepared as described in [12]), EDCxHCl and anhydrous pyridine. The resulting orange suspension was stirred at room temperature to full conversion (1 h) and the orange solution was evaporated to dryness. The residue was dissolved in EtOAc and washed with IN aqueous HC1 (x2) and brine, dried (Na₂SO₄) and evaporated. Pure products were obtained after column chromatography.

Methyl 2-{5-bromo-2-[(15)-l-[(25)-2-{[(tert-butoxy)carbonyl]amino}-3-(3-cyano-2,3-dihydro- lH-inden-5-yl)propanamido]-2,2-dimethylpropyl]-l,3-oxazol-4-yl}-l,3-oxazole-4-carboxylate

(5). Compound was prepared according to General procedure A from ester 3 (1.90 g; 5.52 mmol), LiOHxH₂O (1.16 g; 27.58 mmol; 5 equiv.) in THF/water (1 : 1 v/v; 15 mL) and amine 4 (1.95 g; 5.44 mmol; 1 equiv.), EDC^XHC1 (1.57 g; 8.17 mmol; 1.5 equiv.) in pyridine (15 mL) in 1 h. Product 5 was purified by reverse phase flash chromatography (from 10% to 70% MeCN in 0.01% TFA in water) to afford 2.65 g (73%) of a 1 :1 mixture of diastereomers as a white solid.

’H NMR (400 MHz, CDCh) 8 8.34 (s, 0.5H), 8.32 (s, 0.5H), 7.28 - 7.24 (m, 1H), 7.16 - 7.04 (m, 2H), 6.77 (d, J= 9.4 Hz, 0.5H), 6.68 (d, J= 9.4 Hz, 0.5H), 5.07 (d, J= 9.4 Hz, 0.5H), 5.04 (d, J= 9.4 Hz, 0.5H), 5.05 - 4.95 (m, 1H), 4.34 - 4.26 (m, 1H), 4.09 - 4.02 (m, 1H), 3.94 (s, 3H), 3.15 - 3.00 (m, 2H), 3.01 - 2.91 (m, 1H), 2.88 - 2.75 (m, 1H), 2.58 - 2.41 (m, 1H), 2.38 - 2.23 (m, 1H), 1.43 (s, 9H), 0.97 (s, 4.5H), 0.95 (s, 4.5H). ¹³C NMR (101 MHz, CDCh) 6 170.95, 170.93, 164.8, 161.38, 161.36, 155.7, 154.81, 154.76, 144.14, 144.05, 141.73, 141.68, 138.2, 136.1, 136.0, 134.7, 129.8,

129.6, 128.1, 125.5, 125.24, 125.22, 125.1, 123.12, 123.09, 121.1, 121.0, 80.6, 56.2, 55.8, 55.7, 52.4,

37.4, 37.2, 36.0, 35.8, 34.5, 31.41, 31.39, 31.19, 31.15, 28.4, 26.32, 26.29. HRMS (m/z): C3iH₃5N₅O7Br [M-H]- found: 668.1722. Calculated: 668.1720.

General procedure B (macrocyclization)

Compound 5 and an oven dried K3PO4 were suspended in anhydrous DMSO. Then the suspension was stirred at 65 °C to full conversion (typically 3-6 h) upon which it was cooled to r.t., quenched with aqueous saturated NH4CI and extracted with EtOAc (x2). Combined organic layers were washed with brine, dried (Na2SO4) and evaporated. Pure products were obtained after reverse phase flash column chromatography.

Methyl 2-[(15',65',95)-9-{[(tert-butoxy)carbonyl]amino}-6-tert-butyl-l-cyano-8-oxo-19-oxa-4,7- diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-3-yl]-l,3-oxazole-4-carboxylate

(6). Compound was prepared according to General procedure B from amide 5 (2.65 g; 3.95 mmol) and K3PO4 (4.19 g; 19.76 mmol; 5 equiv.) in DMSO (120 mL) in 5 h. Product 6 was purified by reverse phase flash chromatography (from 10% to 70% MeCN in 0,01% TFA in water) to afford 1.865 g (80%, 99: 1 d.r.) of title compound as a white amorphous solid.

’H NMR (400 MHz, CDCh) 6 8.37 (s, 1H), 7.23 - 7.21 (m, 2H), 7.12 (s, 1H), 5.73 (d, J= 7.8 Hz, 1H), 5.24 (d, J= 9.2 Hz, 1H), 4.81 (d, J= 7.8 Hz, 1H), 3.94 (s, 3H), 3.89 (ddd, J= 12.0, 9.2, 3.2 Hz, 1H), 3.32 - 3.12 (m, 3H), 2.99 - 2.89 (m, 2H), 2.86 (dd, J= 12.4, 3.4 Hz, 1H), 1.43 (s, 9H), 1.00 (s, 9H). ¹³C NMR (101 MHZ, CDCh) 8 171.9, 162.0, 161.4, 155.3, 154.8, 148.9, 144.6, 141.4, 140.9,

135.6, 134.8, 130.3, 129.3, 127.1, 125.5, 118.2, 80.5, 58.3, 57.8, 52.4, 46.4, 38.5, 37.9, 33.5, 31.1,

28.4, 26.6. HRMS (m/z): C31H36N5O7 [M+H]⁺ found: 590.2617. Calculated: 590.2615. [a]_D ²⁰ -129 (c 1.0, CHCh).

General procedure C (cleavage of /V-Boc and introduction of II/VA sidechain) A-Boc protected macrocyle was dissolved in anhydrous DCM and TFA was added. The solution was stirred at room temperature to full conversion (typically 1-2 h). The flask was placed in ice bath and aqueous saturated NaHCCf was added slowly. CO2 evolution! Organic layer was washed with brine, dried (Na2SO4) and evaporated to dryness. The yellow amorphous solid was dissolved in anhydrous DMF and fS')-2-hydroxy-3 -methylbutanoic acid (5-HzVA; 7), EDOHC1 and HOBt were added followed by DIPEA. The solution was stirred at room temperature until full conversion was achieved (typically 1 h). The solution was diluted with aqueous saturated NH4CI and EtOAc. The layers were separated and the organic phase was washed with brine, dried (Na2SO4) and evaporated. Pure products were obtained by reverse phase flash column chromatography.

Methyl 2- [( 15',65',95)-6-ferf-butyl- l-cyano-9- [(25)-2-hydroxy-3-methylbutanamido] -8-oxo- 19- oxa-4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-3-yl]-l,3-oxazole-4- carboxylate (DZA-129). Compound was prepared according to General procedure C from macrocycle 6 (250 mg; 0.42 mmol), TFA (650 pL; 8.48 mmol; 20 equiv.) in DCM (2 mL) in 1 h and 5-HzVA (75 mg; 0.64 mmol; 1.5 equiv.), EDC^XHC1 (163 mg; 0.85 mmol; 2 equiv.), HOBt (172 mg; 1.27 mmol; 3 equiv.), DIPEA (367 pL; 2.12 mmol; 5 equiv.) in DMF (2 mL) in 1 h . Purification by reverse phase flash chromatography (10% to 50% MeCN in 0.01% TFA in water) afforded 180 mg (72%) of the title compound DZA-129 as a white amorphous solid.

’H NMR (400 MHz, MeOD) 5 8.75 (s, 1H), 7.44 - 7.21 (m, 2H), 6.97 (d, J= 1.5 Hz, 1H), 4.74 (s, 1H), 4.54 (dd, J= 11.8, 3.8 Hz, 1H), 3.94 (s, 3H), 3.86 (d, = 3.8 Hz, 1H), 3.29 - 3.10 (m, 3H), 3.05 - 2.80 (m, 3H), 2.09 (sept d, J= 6.9, 3.8 Hz, 1H), 1.01 (s, 9H), 1.00 (d, J= 6.8 Hz, 3H), 0.89 (d, J= 6.8 Hz, 3H). ¹³C NMR (101 MHZ, MeOD) 5 175.8, 173.9, 164.4, 162.7, 156.2, 150.7, 146.8, 142.9, 142.5, 136.8, 135.4, 131.9, 129.6, 127.7, 126.6, 119.3, 76.8, 59.6, 56.2, 52.7, 47.8, 39.2, 39.1, 34.3, 33.2, 31.8, 26.8, 19.5, 16.3. HRMS (m/z): C31H36N5O7 [M+H]⁺ found: 590.2619. Calculated: 590.2615. [a]_D ²⁰ -182 (c 1.0, MeOH).

The synthesis of diazonamide A analogs DZA-135, 139, 164-165 is shown in Scheme 2.

Ester analogs DZA-135, DZA-139, DZA-164 and DZA-165 were synthesized from DZA-129. Accordingly, ethyl ester (DZA-139) and benzyl ester (DZA-135) were prepared from methyl ester DZA-129 by Lewis acid-catalyzed transesterification reaction in the presence of EtOH or BnOH, respectively. Macrocycles DZA-164 and DZA-165 were obtained by initial hydrolysis of DZA-129 in the presence of LiOHxH2O and subsequent alkylation of the resulting acid with iodomethyl pivalate to give ester DZA-164 or with 1-bromoethyl acetate to give analog DZA-165 (Scheme 2).

Scheme 2. Synthesis of diazonamide A analogs DZA-135, DZA-139, DZA-164, and DZA-165

Benzyl 2- [( 15',65',95)-6-tert-butyl- l-cyano-9- [(25)-2-hydroxy-3-methylbutanamido] -8-oxo- 19- oxa-4,7-diazatetracyclo[9.5.2.1², .O¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-3-yl]-l,3-oxazole-4- carboxylate (DZA-135). To methyl ester DZA-129 (50 mg; 0.085 mmol) in toluene (1 mL) was added benzyl alcohol (350 pL; 3.39 mmol; 40 equiv.) followed by the addition of Ti(O/Pr)4 (25 pL; 0.085 mmol; 1 equiv.). The resulting yellow solution was stirred at 100°C in mineral oil bath for 2 h. Then the vial was cooled to room temperature and diluted with EtOAc. The resulting solution was washed with aqueous IN HC1 (x2), then brine. The organic layer was dried (Na2SO4) and evaporated. The resulting yellow oil was purified with reverse phase flash chromatography (10% to 70% MeCN in water) to give benzyl ester DZA-135 (35 mg; 62%) as a white amorphous solid.

’H NMR (400 MHz, MeOD) 5 8.77 (s, 1H), 7.51 - 7.46 (m, 2H), 7.43 - 7.29 (m, 5H), 6.97 (s, 1H), 5.40 (s, 2H), 4.74 (s, 1H), 4.54 (dd, J = 11.8, 3.8 Hz, 1H), 3.86 (d, J = 3.8 Hz, 1H), 3.28 - 3.11 (m, 3H), 3.01 - 2.91 (m, 2H), 2.88 (d, J = 12.4, 3.8 Hz, 1H), 2.08 (sept d, J= 6.9, 3.8 Hz, 1H), 1.01 (s, 9H), 1.00 (d, .7= 6.9 Hz, 3H), 0.89 (d, = 6.9 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.8, 173.9, 164.4, 162.0, 156.3, 150.7, 147.0, 142.9, 142.5, 137.1, 136.8, 135.5, 131.9, 129.6, 129.6, 129.4, 129.4, 127.7, 126.6, 119.4, 76.8, 67.9, 59.6, 56.3, 47.8, 39.2, 39.1, 34.3, 33.2, 31.8, 26.7, 19.5, 16.3. HRMS (m/z): C37H40N5O7 [M+H]⁺ found: 666.2934. Calculated: 666.2928. [a]_D ²⁰ -128 (c 1.0, MeOH).

Ethyl 2- [( 15.6.S.9.S')-6-/cr/-bii t I- l-cyano-9- [(25)-2-hydroxy-3-methylbutanamido] -8-oxo- 19- oxa-4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-3-yl]-l,3-oxazole-4- carboxylate (DZA-139). To methyl ester DZA-129 (38 mg; 0.064 mmol) in toluene (0.5 mL) was added ethanol (188 pL; 3.22 mmol; 50 equiv.) followed by the addition of Ti(O/Pr)4 (19 pL; 0.065 mmol; 1 equiv.). The resulting yellow solution was stirred at 100°C in mineral oil bath for 20 h. Then the vial was cooled to room temperature and diluted with EtOAc. The resulting solution was washed with aqueous IN HC1 (x2), then brine. The organic layer was dried (Na2SO4) and evaporated. The resulting yellow oil was purified with reverse phase flash chromatography (10% to 70% MeCN in water) to give ethyl ester DZA-139 (24 mg; 62%) as a white amorphous solid.

’H NMR (400 MHz, MeOD) 5 8.74 (s, 1H), 8.37 (d, J = 7.6 Hz, 1H), 7.42 - 7.26 (m, 2H), 6.97 (s, 1H), 4.78 - 4.71 (m, 1H), 4.54 (dd, J= 11.8, 3.8 Hz, 1H), 4.41 (q, J= 7.1 Hz, 2H), 3.86 (d, J= 3.8 Hz, 1H), 3.29 - 3.10 (m, 3H), 3.03 - 2.91 (m, 2H), 2.88 (dd, J= 12.2, 3.8 Hz, 1H), 2.09 (sept d, J = 6.9, 3.8 Hz, 1H), 1.40 (t, J= lA z, 3H), 1.01 (s, 9H), 1.00 (d, J= 6.8 Hz, 3H), 0.89 (d, J= 6.8 Hz, 3H). ¹³C NMR (101 MHZ, MeOD) 5 175.8, 173.9, 164.4, 162.3, 156.2, 150.6, 146.7, 142.9, 142.5,

136.8, 135.7, 131.9, 129.6, 127.7, 126.6, 119.4, 76.8, 62.5, 59.6, 56.3, 47.8, 39.3, 39.2, 34.3, 33.2,

31.8, 26.7, 19.5, 16.3, 14.6. HRMS (m/z): C32H38N5O7 [M+H]⁺ found: 604.2783. Calculated: 604.2771. [a]_D ²⁰ -147 (c 1.0, MeOH).

{2- [( LS.6.S.9.S)-6-/‘cr/‘- But y I- l-cyano-9- | ( 2.S’)- 2- hydro xy-3-methylbutanamido] -8-oxo- 19-oxa-4,7 - diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-3-yl]-l,3-oxazole-4- carbonyloxy}methyl 2,2-dimethylpropanoate (DZA-164). Methyl ester DZA-129 (45 mg; 0.076 mmol) was dissolved in THF (0.5 mL). Separately solid LiOHxH2O (10 mg; 0.23 mmol; 3 equiv.) was dissolved in water (0.2 mL) and added to the reaction mixture. Emulsion was stirred at room temperature for 30 min, then aqueous IN HC1 was added. The resulting mixture was extracted with EtOAc (x2), combined organic layers were washed with brine, dried (Na2SO4) and evaporated. The crude carboxylic acid was used in the next step without additional purification. The crude acid was dissolved in dry DMF (0.5 mL), then K2CO3 (21 mg; 0.15 mmol; 2 equiv.) was added at room temperature followed by iodomethyl pivalate (43 pL; 0.15 mmol; 2 equiv.). The suspension was stirred for 1 h, then it was diluted with EtOAc and washed with aqueous saturated NH4CI (x2). Organic layer was washed with brine, dried (Na2SO4), evaporated. The resulting dark brown oil was purified with reverse phase flash chromatography (10% to 70% MeCN in 0.01% TFA in water) to give ester DZA-164 (25 mg; 48%) as a white amorphous solid.

’H NMR (400 MHz, MeOD) 5 8.83 (s, 1H), 8.36 (d, J = 7.6 Hz, 1H), 7.38 - 7.29 (m, 2H), 7.98 (s, 1H), 6.01 (d, J = 7.6 Hz, 1H), 6.00 (d, J = 7.6 Hz, 1H), 4.79 - 4.71 (m, 1H), 4.54 (dd, J = 11.8, 3.8 Hz, 1H), 3.86 (d, J = 3.8 Hz, 1H), 3.29 - 3.09 (m, 3H), 3.04 - 2.92 (m, 2H), 2.89 (dd, J = 12.4, 3.8 Hz, 1H), 2.08 (sept d, J= 6.9, 3.8 Hz, 1H), 1.23 (s, 9H), 1.01 (s, 9H), 1.00 (d, J= 6.8 Hz, 3H), 0.89 (d, J = 6.8 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 178.3, 175.8, 174.0, 164.4, 160.7, 156.5, 150.8, 147.8, 142.9, 142.4, 136.8, 134.6, 131.9, 129.6, 127.6, 126.6, 119.3, 81.1, 76.8, 59.7, 56.3, 47.8, 39.8, 39.22, 39.15, 34.3, 33.2, 31.8, 27.2, 26.7, 19.5, 16.3. HRMS (m/z): C36H44N5O9 [M+H]⁺ found: 690.3152. Calculated: 690.3139. [a]_D ²⁰ -161 (c 1.0, MeOH). l-{2-[(15',65',95)-6-tert-Butyl-l-cyano-9-[(25)-2-hydroxy-3-methylbutanamido]-8-oxo-19-oxa- 4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-3-yl]-l,3-oxazole-4- carbonyloxy}ethyl acetate (DZA-165). Methyl ester DZA-129 (130 mg; 0.22 mmol) was dissolved in THF (1 mL). Separately solid LiOHxIHkO (28 mg; 0.66 mmol; 3 equiv.) was dissolved in water (0.5 mL) and added to the reaction mixture. Emulsion was stirred at room temperature for 30 min, then aqueous IN HC1 was added. The resulting mixture was extracted with EtOAc (x2), combined organic layers were washed with brine, dried (Na2SO4) and evaporated. The crude carboxylic acid was used in the next step without additional purification. The crude acid was dissolved in dry DMF (1 mL), then K2CO3 (61 mg; 0.44 mmol; 2 equiv.) was added at room temperature followed by 1- bromoethylacetate (74 pL; 0.44 mmol; 2 equiv.). The suspension was stirred for 1 h, then it was diluted with EtOAc and washed with aqueous saturated NFLCl (x2). Organic layer was washed with brine, dried (Na2SO4), evaporated. The resulting dark brown oil was purified with reverse phase flash chromatography (10% to 70% MeCN in 0.01% TFA in water) to give ester DZA-165 (62 mg; 43%) as a white amorphous solid.

¹H NMR (400 MHz, MeOD) 5 8.81 (d, J= 1.8 Hz, 1H), 8.36 (d, J= 7.6 Hz, 1H), 7.40 - 7.22 (m, 2H), 7.09 (q, .7= 5.4 Hz, 1H), 6.98 (s, 1H), 4.79 - 4.69 (m, 1H), 4.54 (dd, J= 11.8, 3.8 Hz, 1H), 3.86 (d, J = 3.8 Hz, 1H), 3.30 - 3.10 (m, 3H), 3.02 - 2.91 (m, 2H), 5 2.89 (dd, J= 12.5, 3.8 Hz, 1H), 2.13 - 2.04 (m, 1H), 2.09 (s, 3H), 1.61 (d, J= 5.4 Hz, 3H), 1.01 (s, 9H), 1.00 (d, J= 6.8 Hz, 3H), 0.89 (d, J = 6.8 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.8, 174.0, 173.9, 170.6, 164.4, 160.1, 156.4, 150.8, 147.6, 142.9, 142.4, 136.8, 134.9, 131.9, 129.6, 127.6, 126.6, 119.4, 90.4, 76.8, 59.6, 56.3, 47.8, 39.2, 34.3, 33.2, 31.8, 26.8, 20.7, 19.7, 19.5, 16.3. HRMS (m/z): C₃4H₃9N₅O9Na [M+Na]⁺ found: 684.2657. Calculated: 684.2645.

The synthesis of diazonamide A analog DZA-143 is shown in Scheme 3.

Accordingly, the crude acid 8 (prepared as described in [12]) was reacted with L-serine tert-butyl ester hydrochloride in the presence of EDC hydrochloride in pyridine to furnish amide 9. The cyclization of 9 into bisoxazole 10 was accomplished by DAST reagent in the presence ofDBU and BrCCh. The final step of the synthesis was the cleavage of N-Boc protecting group to afford the building block 11. Accordingly, ester hydrolysis in 12 was followed by amide coupling with corresponding amine 11 giving amide 13. The key macrocyclization step in presence of K3PO4 furnished macrocycle 14 with diastereomeric ratio 97:3. After -Boc cleavage with MsOH the resulting amine was coupled with (5)-2-hydroxy-3 -methylbutanoic acid (5-HzVA; 7) to give analog DZA-143 (Scheme 3).

Scheme 3. Synthesis of diazonamide A analog DZA-143 tert- Butyl (25)-2-({5-bromo-2-[(15)-l-{[(tert-butoxy)carbonyl]amino}-2,2-dimethylpropyl]-l,3- oxazol-4-yl}formamido)-3-hydroxypropanoate (9). Carboxylic acid 8 (500 mg; 1.33 mmol) was dissolved in dry pyridine (5 mL), EDC^XHC1 (356 mg; 1.86 mmol; 1.4 equiv.) and L-SerOtBu (328 mg; 1.66 mmol; 1.25 equiv.) were added at room temperature. The resulting solution was stirred at room temperature for 1 h, then pyridine was evaporated and the resulting orange oil was redissolved in EtOAc and washed with aqueous IN HC1 (x2). Combined organic layers were washed with brine, dried (Na2SC>4) and evaporated. The resulting orange oil was purified with direct phase flash chromatography (20% to 40% EtOAc in hexanes) to give amide 9 (310 mg; 45%) as a yellow oil.

‘H NMR (400 MHz, CDCh) 8 7.72 (d, J= 7.2 Hz, 1H), 5.19 (d, J= 9.9 Hz, 1H), 4.71 - 4.64 (m, 2H), 4.06 - 3.97 (m, 2H), 1.50 (s, 9H), 1.44 (s, 9H), 0.98 (s, 9H). ¹³C NMR (101 MHz, CDCh) 6 169.1, 164.2, 160.2, 155.4, 131.3, 125.1, 83.3, 80.5, 64.1, 57.5, 55.6, 35.7, 28.4, 28.2, 26.3. HRMS (m/z): C2iH₃₄N₃O₇NaBr [M+Na]⁺ found: 542.1487. Calculated: 542.1478. [a]_D ²⁰ -7 (c 1.0, CHC1₃). tert-Butyl 2-{5-bromo-2-[(15)-l-{[(tert-butoxy)carbonyl]amino}-2,2-dimethylpropyl]-l,3- oxazol-4-yl}-l,3-oxazole-4-carboxylate (10). To alcohol 9 (800 mg; 1.54 mmol) in dry DCM (7 mL) DAST (207 pL; 1.69 mmol; 1.1 equiv.) was added dropwise after the flask was cooled to -78°C. The orange solution was gradually warmed to 0 °C and K2CO3 (850 mg; 6.15 mmol; 4 equiv.) was added in one portion. The resulting suspension was stirred for 30 minutes at the same temperature. Then the flask was shielded from the light with aluminum foil and neatBrCCh (378 pL; 3.84 mmol; 2.5 equiv.) was added followed by DBU (574 pL; 3.84 mmol; 2.5 equiv.). The dark red reaction mixture was stirred at room temperature for 20 h. The dark red suspension was quenched by the addition of aqueous 4N HC1 (Caution! CO2 evolution) and extracted with EtOAc (x3). Organic layers were combined, washed with water, brine, dried (Na2SO4), evaporated. The resulting dark oil was purified with direct phase flash chromatography (0% to 20% EtOAc in hexanes) to give bioxazole 10 (420 mg; 55%) as colourless oil.

³H NMR (400 MHz, CDCh) 5 8.18 (s, 1H), 5.34 (d, J = 9.9 Hz, 1H), 4.74 (d, J = 9.9 Hz, 1H), 1.59 (s, 9H), 1.42 (s, 9H), 1.00 (s, 9H). ¹³C NMR (101 MHz, CDCh) 8 165.8, 160.1, 155.4, 154.7, 143.4, 136.1, 128.2, 122.9, 82.6, 80.3, 57.5, 36.0, 28.4, 28.3, 26.3. HRMS (m/z): C₂iH₃₀N₃O₆NaBr [M+Na]⁺ found: 522.1218. Calculated: 522.1216. [a]_D ²⁰ -34 (c 1.0, CHCh). tert- Butyl 2-{5-bromo-2-[(15)-l-[(25)-2-{[(tert-butoxy)carbonyl]amino}-3-(3-cyano-2,3- dihydro-lH-inden-5-yl)propanamido]-2,2-dimethylpropyl]-l,3-oxazol-4-yl}-l,3-oxazole-4- carboxylate (13). Compound was prepared according to General procedure C from ester 12 (290 mg; 0.84 mmol), LiOHxH2O (177 mg; 4.21 mmol; 5 equiv.) in THF/water (1 : 1 v/v; 3 mL) and amine 11 (337 mg; 0.84 mmol; 1 equiv.), EDC^XHC1 (242 mg; 1.26 mmol; 1.5 equiv.) in pyridine (6 mL) in 1 h. Product 13 was purified by reverse phase flash chromatography (from 10% to 70% MeCN in 0,01% TFA in water) to afford 13 (230 mg, 49%) of a 1 : 1 mixture of diastereomers as a white amorphous solid.

’H NMR (400 MHz, CDCh) 5 8.20 (s, 0.5H), 8.19 (s, 0.5H), 7.27 (s, 1H), 7.14 - 7.05 (m, 2H), 6.73 (d, J= 8.4 Hz, 0.5H), 6.64 (d, J= 8.4 Hz, 0.5H), 5.06 (t, J= 9.6 Hz, 1H), 5.07 - 4.96 (m, 1H), 4.34 - 4.26 (m, 1H), 4.08 - 4.03 (m, 1H), 3.14 - 3.02 (m, 2H), 3.01 - 2.92 (m, 1H), 2.87 - 2.74 (m, 1H), 2.55 - 2.44 (m, 1H), 2.37 - 2.25 (m, 1H), 1.60 (s, 9H), 1.43 (s, 9H), 0.96 (s, 4.5H), 0.95 (s, 4.5H). ¹³C NMR (101 MHz, CDCh) 6 171.0, 170.9, 164.7, 160.1, 160.0, 155.7, 155.6, 154.52, 154.47, 143.43, 143.36, 141.7, 138.2, 136.1, 136.0, 129.8, 129.6, 128.2, 125.5, 125.2, 125.1, 123.0, 121.1, 121.0, 82.7, 80.6, 56.2, 55.8, 55.7, 37.5, 37.3, 36.0, 35.9, 34.5, 31.4, 31.2, 28.4, 28.3, 26.3. HRMS (m/z): C₃₄H₄₂N₅O₇NaBr [M+Na]⁺ found: 734.2167. Calculated: 734.2165. tert-Butyl 2-[(15',65',95)-9-{[(tert-butoxy)carbonyl]amino}-6-tert-butyl-l-cyano-8-oxo-19-oxa- 4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-3-yl]-l,3-oxazole-4- carboxylate (14). Compound was prepared according to General procedure B from amide 13 (220 mg; 0.31 mmol) and K₃PO₄ (328 mg; 1.54 mmol; 5 equiv.) in DMF (10 mL) in 4 h. Product 14 was purified by reverse phase flash chromatography (from 10% to 70% MeCN in 0,01% TFA in water) to afford 150 mg (77%) of a single diastereomer 14 as a white amorphous solid.

’H NMR (400 MHz, CDCh) 8 8.25 (s, 1H), 7.24 - 7.19 (m, 2H), 7.15 (s, 1H), 5.69 (d, J = 7.9 Hz, 1H), 5.22 (d, J= 9.2 Hz, 1H), 4.83 (d, J= 7.9 Hz, 1H), 3.89 (ddd, J= 12.3, 9.2, 3.3 Hz, 1H), 3.32 - 3.21 (m, 2H), 3.19 - 3.11 (m, 1H), 3.03 - 2.91 (m, 2H), 2.90 - 2.83 (m, 1H), 1.60 (s, 9H), 1.43 (s, 9H), 1.01 (s, 9H). ¹³C NMR (101 MHZ, CDCh) 6 171.9, 161.9, 160.1, 155.3, 154.6, 148.6, 144.0, 141.4, 140.9, 136.2, 135.5, 130.3, 129.4, 127.2, 125.5, 118.3, 82.5, 80.5, 58.3, 57.7, 46.4, 38.2, 37.9,

33.5, 31.1, 28.4, 28.4, 26.6. HRMS (m/z): C34H42N5O7 [M+H]⁺ found: 632.3085. Calculated: 632.3084. [a]_D ²⁰ -115 (c 1.0, CHCh). tert- Butyl 2-[(15',65',95)-6-tert-butyl-l-cyano-9-[(25)-2-hydroxy-3-methylbutanamido]-8-oxo-

19-oxa-4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-3-yl]-l,3-oxazole-4- carboxylate (DZA-143). Compound was prepared according to General procedure C from macrocycle 14 (120 mg; 0.19 mmol), MsOH (43 pL; 0.66 mmol; 3.5 equiv.) in DCM/tBuOAc (4:1 v/v; 0.5 mL) in 2 h and 5-HzVA (34 mg; 0.29 mmol; 1.5 equiv.), EDC^XHC1 (73 mg; 0.38 mmol; 2 equiv.), HOBt (77 mg; 0.57 mmol; 3 equiv.), DIPEA (164 pL; 0.95 mmol; 5 equiv.) in DMF (2 mL) in 1 h . Product DZA-143 was purified by reverse phase flash chromatography (10% to 95% MeCN in water) to afford 21 mg (20%) as a white amorphous solid.

^XH NMR (400 MHz, MeOD) 6 8.58 (s, 1H), 7.37 - 7.30 (m, 2H), 7.00 (s, 1H), 4.75 (s, 1H), 4.52 (dd, J = 11.8, 3.8 Hz, 1H), 3.86 (d, J= 3.8 Hz, 1H), 3.29 - 3.08 (m, 3H), 3.02 - 2.83 (m, 3H), 2.09 (sept d, J= 6.8, 3.8 Hz, 1H), 1.61 (s, 9H), 1.01 (s, 9H), 1.00 (d, J= 6.8 Hz, 3H), 0.90 (d, J= 6.8 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.8, 173.9, 164.3, 161.6, 156.1, 150.6, 146.2, 142.9, 142.5, 136.89, 136.87, 131.9, 129.7, 127.9, 126.5, 119.5, 83.7, 77.0, 59.7, 56.4, 47.8, 39.3, 39.2, 34.3, 33.2, 31.8,

28.5, 26.8, 19.4, 16.4. HRMS (m/z): C34H₄iN₅O7Na [M+Na]⁺ found: 654.2922. Calculated: 654.2904. [a]_D ²⁰ -144 (c 1.0, MeOH).

The synthesis of diazonamide A analog DZA-163 is shown in Scheme 4.

Accordingly, ketone 15 was transformed into nitrile 16 in the presence of TosMIC and KO/Bu, then the resulting nitrile 16 was reacted with freshly prepared organozinc species 2 in Pd-catalyzed Negishi cross-coupling. Ester hydrolysis in 17 was followed by amide coupling with corresponding amine 4 giving amide 18. The key macrocyclization step in presence of K3PO4 furnished macrocycle 19 with diastereomeric ratio 98:2. After A-Boc cleavage with TFA the resulting amine was coupled with (5)- 2-hydroxy-3 -methylbutanoic acid fS-H/VA; 7) to give analog DZA-163 (Scheme 4).

Scheme 4. Synthesis of diazonamide A analog DZA-163

6- Bromo-4-fluoro-2,3-dihyd ro- 1 //-indene- 1 -carbonit rile (16). Ketone 15 (650 mg; 2.84 mmol) and TosMIC (665 mg; 3.41 mmol; 1.2 equiv.) were dissolved in dry THF (7 mL) and cooled to -78 °C. EtOH (200 pL; 3.41 mmol; 1.2 equiv.) and Z-BuOK (446 mg; 3.97 mmol; 1.40 equiv.) were added sequentially and stirring was continued for 2 h while the cold bath was allowed to warm to room temperature gradually. The cold bath was removed and the mixture was stirred at room temperature for 20 h. Dark red thick solution. The solution was diluted with water, then IN HC1 was added and the mixture was extracted with EtOAc (x2). Organic layers were combined, washed with brine, dried (Na2SO4) and evaporated. Dark red solid. The residue was purified with column chromatography (10% EtOAc in hexanes) to give the product 16 (310 mg, 46%) as yellowish oil.

'H NMR (400 MHz, CDCh) 8 7.38 (s, 1H), 7.16 (d, J = 8.2 Hz, 1H), 4.13 (t, J = 8.2 Hz, 1H), 3.15 - 3.06 (m, 1H), 2.98 - 2.84 (m, 1H), 2.70 - 2.55 (m, 1H), 2.47 - 2.37 (m, 1H). ¹³C NMR (101 MHz, CDCh) 6 158.8 (d, J= 252.6 Hz), 142.4 (d, J= 6.8 Hz), 128.8 (d, J= 19.8 Hz), 123.6 (d, J= 3.8 Hz), 121.3 (d, J= 7.9 Hz), 119.9 (s), 119.1 (d, J = 23.7 Hz), 34.7 (d, J= 2.3 Hz), 31.2, 27.4. HRMS (m/z): CioH₆NBrF [M+H]⁺ found: 237.9662. Calculated: 237.9668.

Methyl (25)-2-{[(tert-butoxy)carbonyl]amino}-3-(3-cyano-7-fluoro-2,3-dihydro-lE/-inden-5- yl)propanoate (17). Aryl bromide 15 (130 mg; 0.54 mmol), Pd2(dba)a (7 mg; 0.008 mmol; 0.02 equiv.) and SPhos (9 mg; 0.022 mmol; 0.04 equiv.) were dissolved in anhydrous DMF (1 mL) under argon atmosphere and freshly prepared solution of alkyl zinc iodide 2 (0.5 M in DMF; 2.2 mL; 1.08 mmol; 2.0 equiv.) (prepared as described in [11]) was added. The dark red solution was stirred at 65 °C for 3 h. Then the solution was cooled to room temperature and aqueous saturated NH4CI and EtOAc were added. Layers were separated and EtOAc was washed with IN HC1 aqueous solution and brine, dried (Na2SO4) and evaporated to dryness. Product 17 was purified by column chromatography on silica (30% EtOAc in hexanes) to afford 65 mg (33%; dr ~1:1) as a orange oil that solidifies upon standing.

’H NMR (400 MHz, MeOD) 5 7.11 (s, 0.5H), 7.09 (s, 0.5H), 6.94 - 6.88 (m, 1H), 4.44 - 4.26 (m, 2H), 3.72 (s 1.5H), 3.70 (s, 1.5H), 3.19 - 3.10 (m, 1H), 3.11 - 3.02 (m, 1H), 3.00 - 2.84 (m, 2H), 2.68 - 2.58 (m, 1H), 2.39 - 2.25 (m, 1H), 1.39 (s, 4.5H), 1.37 (s, 4.5H). ¹³C-{¹⁹F} NMR (151 MHz, MeOD) 5 173.8, 173.7, 160.3, 160.2, 157.7, 142.9, 140.9, 129.04, 128.97, 122.02, 121.96, 121.84, 121.81, 117.0, 116.9, 80.7, 56.4, 56.2, 52.8, 52.7, 38.2, 38.1, 35.5, 32.4, 28.64, 28.60, 28.0. HRMS (m/z): Ci9H23N₂O₄FNa [M+Na]⁺ found: 385.1536. Calculated: 385.1540.

Methyl 2-{5-bromo-2-[(15)-l-[(25)-2-{[(tert-butoxy)carbonyl]amino}-3-(3-cyano-7-fluoro-2,3- dihydro-lH-inden-5-yl)propanamido]-2,2-dimethylpropyl]-l,3-oxazol-4-yl}-l,3-oxazole-4- carboxylate (18). Compound was prepared according to General procedure A from ester 17 (50 mg; 0.14 mmol), LiOHxH₂O (29 mg; 0.69 mmol; 5 equiv.) in THF/water (1 : 1 v/v; 1 mL) and amine 4 (49 mg; 0.14 mmol; 1 equiv.), EDC^XHC1 (40 mg; 0.21 mmol; 1.5 equiv.) in pyridine (1 mL) in 1 h. Product was purified by reverse phase flash chromatography (from 10% to 70% MeCN in 0,01% TFA in water) to afford 45 mg (47%) of a 1 : 1 mixture of diastereomers of 18 as a yellow amorphous solid.

’H NMR (600 MHz, MeOD) 5 8.69 (s, 1H), 7.09 (s, 1H), 6.84 (d, J= 10.0 Hz, 0.5H), 6.82 (d, J= 9.8 Hz, 0.5H), 5.02 (d, J= 9.4 Hz, 1H), 4.44 - 4.37 (m, 1H), 4.27 (dd, J= 8.1, 8.1 Hz, 0.5H), 4.22 (dd, J = 8.1, 8.1 Hz, 0.5H), 3.93 (s, 1.5H), 3.92 (s, 1.5H), 3.03 - 2.83 (m, 3H), 2.81 - 2.68 (m, 1H), 2.56 - 2.48 (m, 1H), 2.28 - 2.19 (m, 1H), 1.41 (s, 4.5H), 1.40 (s, 4.5H), 1.02 (s, 4.5H), 1.01 (s, 4.5H). ¹³C- {¹⁹F} NMR (151 MHz, MeOD) 5 174.0, 173.9, 166.1, 162.6, 160.1, 157.5, 156.3, 156.2, 146.3, 142.74, 142.67, 140.7, 140.6, 135.34, 135.30, 128.9, 128.73, 128.7, 124.9, 124.8, 122.2, 122.1, 121.73, 121.68, 116.99, 116.95, 80.7, 57.1, 57.0, 52.7, 38.6, 38.5, 36.7, 36.6, 35.5, 32.29, 32.25, 28.6, 27.92, 27.88, 26.6. HRMS (m/z): CaiHasNsO FNaBr [M+Na]⁺ found: 710.1603. Calculated: 710.1602.

Methyl 2-[(15',65',95)-9-{[(tert-butoxy)carbonyl]amino}-6-tert-butyl-l-cyano-13-fluoro-8-oxo- 19-oxa-4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-3-yl]-l,3-oxazole-4- carboxylate (19). Compound was prepared according to General procedure B from amide 18 (46 mg; 0.067 mmol) and K3PO4 (71 mg; 0.34 mmol; 5 equiv.) in DMF (2 mL) in 8 h. Product 19 was purified by reverse phase flash chromatography (from 10% to 70% MeCN in 0,01% TFA in water) to afford 21 mg (52%; dr 98:2) of a single diastereomer as a white amorphous solid.

’H NMR (400 MHz, CDCh) 8 8.68 (s, 1H), 7.57 (s, 1H), 7.28 - 7.25 (m, 2H), 6.16 (d, J= 7.7 Hz, 1H), 5.51 (d, J = 9.3 Hz, 1H), 5. 11 (d, J= 7.7 Hz, 1H), 4.25 (s, 3H), 4.24 - 4.19 (m, 1H), 3.62 - 3.45 (m, 3H), 3.40 - 3.25 (m, 2H), 3.13 (dd, J= 12.3, 3.3 Hz, 1H), 1.74 (s, 9H), 1.33 (s, 9H). ¹³C NMR (101 MHz, CDCh) 8 171.6, 162.1, 161.3, 159.1 (d, J= 250.4 Hz), 155.3, 154.7, 148.1, 144.6, 143.6 (d, J = 6.5 Hz), 138.2 (d, J= 6.1 Hz), 134.8, 128.2 (d, J = 20.6 Hz), 127.3, 125.3 (d, J = 3.6 Hz), 117.7, 116.3 (d, J = 20.3 Hz), 80.7, 58.0, 57.9, 52.4, 46.6, 38.3, 37.7, 33.5, 28.4, 27.6, 26.6. HRMS (m/z): C₃iH₃4N₅O7FNa [M+Na]⁺ found: 630.2361. Calculated: 630.2340. [a]_D ²⁰ -121 (c 1.0, CHC1₃).

Methyl 2-[(15',65',95)-6-ZerZ-butyl-l-cyano-13-fluoro-9-[(25)-2-hydroxy-3-methylbutanamido]- 8-oxo-19-oxa-4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-3-yl]-l,3- oxazole-4-carboxylate (DZA-163). Compound was prepared according to General procedure C from macrocycle 19 (20 mg; 0.033 mmol), TFA (50 pL; 0.66 mmol; 20 equiv.) in DCM (0.5 mL) in 1 h and 5-HzVA (6 mg; 0.049 mmol; 1.5 equiv.), EDC^XHC1 (19 mg; 0.099 mmol; 3 equiv.), HOBt (13 mg; 0.099 mmol; 3 equiv.), DIPEA (29 pL; 0.17 mmol; 5 equiv.) in DMF (0.5 mL) in 1 h . Product DZA-163 was purified by reverse phase flash chromatography (10% to 70% MeCN in 0,01% TFA in water) to afford 15 mg (75%) as a white amorphous solid.

^XH NMR (400 MHz, MeOD) 6 8.75 (s, 1H), 7.15 (dd, J= 9.8, 1.3 Hz, 1H), 6.83 (d, J= 1.3 Hz, 1H), 4.74 (s, 1H), 4.57 (dd, J = 11.8, 3.8 Hz, 1H), 3.94 (s, 3H), 3.86 (d, J = 3.8 Hz, 1H), 3.28 - 3.13 (m, 3H), 3.12 - 2.97 (m, 2H), 2.89 (dd, J= 12.4, 3.8 Hz, 1H), 2.08 (sept d, J= 6.9, 3.8 Hz, 1H), 1.02 (s, 9H), 0.99 (d, J= 6.8 Hz, 3H), 0.89 (d, J= 6.8 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 6 175.9, 173.7, 164.4, 162.7, 160.5 (d, J = 248.9 Hz), 156.1, 149.8, 146.9, 145.3 (d, J = 6.4 Hz), 139.8 (d, J = 6.6 Hz), 135.4, 129.4 (d, J = 20.6 Hz), 127.9, 125.8 (d, J= 3.2 Hz), 118.9, 117.9 (d, J= 21.0 Hz), 76.8, 59.6, 56.1, 52.7, 48.0, 38.9, 34.4, 33.2, 30.8, 28.0, 26.7, 19.5, 16.4. HRMS (m/z): C₃IH₃5N₅O₇F [M+H]⁺ found: 608.2537. Calculated: 608.2521. [a]_D ²⁰ -154 (c 1.0, MeOH).

The synthesis of diazonamide A analogs DZA-140-141, 144-145 is shown in Scheme 5.

Accordingly, ketones 20a-c were converted into corresponding nitriles 21a-c in the presence of TosMIC and KOtBu. Then nitriles 21a-c were reacted with freshly prepared organozinc species 2 in Pd-catalyzed Negishi cross-coupling. Ester hydrolysis in 22a-c was followed by amide coupling with corresponding amine 4 giving amides 23a-c. The key macrocyclization step in presence of K3PO4 furnished macrocycles 24a-c with diastereomeric ratio 96:4 to 99: 1. After N-Boc cleavage with TFA the resulting amine was coupled with (5)-2-hy dr oxy-3 -methylbutanoic acid fS’-H/VA; 7) to give analogs DZA-140, 141, 144. Analog DZA-145 was furnished from DZA-144 after oxidation with /77CPBA (Scheme 5).

Scheme 5. Synthesis of diazonamide A analogs DZA-140-141, 144-145,

7-Bromo-l,2,3,4-tetrahydronaphthalene-l-carbonitrile (21a). Ketone 20a (1 g; 4.44 mmol) and TosMIC (1.04 g; 5.33 mmol; 1.2 equiv.) were dissolved in dry THF (10 mL) and cooled to -78 °C. EtOH (311 pL; 5.33 mmol; 1.2 equiv.) and t-BuOK (698 mg; 6.22 mmol; 1.4 equiv.) were added sequentially and stirring was continued for 2 h while the cold bath was allowed to warm to room temperature gradually. The cold bath was removed and the mixture was stirred at room temperature for 20 h. Dark red thick solution. The solution was diluted with water, then IN HC1 was added and the mixture was extracted with EtOAc (x2). Organic layers were combined, washed with brine, dried (Na2SO4) and evaporated. Dark red solid. The residue was purified with column chromatography (10% EtOAc in hexanes) to give the product 21a (786 mg, 75%) as white solid.

’H NMR (400 MHz, CDCh) 8 7.51 (d, J= 2.1 Hz, 1H), 7.34 (dd, J = 8.2, 2.1 Hz, 1H), 7.00 (d, J = 8.2 Hz, 1H), 3.94 (dd, J= 6.3, 6.3 Hz, 1H), 2.86 - 2.65 (m, 2H), 2.19 - 2.09 (m, 2H), 2.09 - 1.97 (m, 1H), 1.90 - 1.78 (m, 1H). ¹³C NMR (101 MHz, CDCh) 6 135.5, 132.0, 131.7, 131.5, 131.3, 121.2, 120.0, 30.7, 28.1, 27.2, 20.7. HRMS (m/z): CnH₉NBr [M-H]’ found: 233.9919. Calculated: 233.9918. 6-Bromo-3.4-dihydro-2//- 1 -benzopyran-4-carbonitrile (21b). Ketone 20b (1 g; 4.40 mmol) and TosMIC (1.03 g; 5.29 mmol; 1.2 equiv.) were dissolved in dry DCM (10 mL) and cooled to -78 °C. EtOH (310 pL; 5.30 mmol; 1.2 equiv.) and t-BuOK (692 mg; 6.17 mmol; 1.4 equiv.) were added sequentially and stirring was continued for 2 h while the cold bath was allowed to warm to room temperature gradually. The cold bath was removed and the mixture was stirred at room temperature for 20 h. Dark red thick solution. The solution was diluted with water, then IN HC1 was added and the mixture was extracted with EtOAc (x2). Organic layers were combined, washed with brine, dried (Na2SO4) and evaporated. Dark red solid. The residue was purified with column chromatography (10% EtOAc in hexanes) to give the product 21b (810 mg, 77%) as white solid.

’H NMR (400 MHz, CDCh) 8 7.42 (dd, J = 2.4, 0.8 Hz, 1H), 7.32 (ddd, J = 8.7, 2.4, 0.8 Hz, 1H), 6.75 (d, J = 8.8 Hz, 1H), 4.36 - 4.30 (m, 1H), 4.28 - 4.17 (m, 1H), 4.00 (t, J = 5.9 Hz, 1H), 2.35 - 2.29 (m, 2H). ¹³C NMR (101 MHz, CDCh) 6 153.3, 133.1, 132.0, 120.0, 119.7, 117.2, 113.2, 63.8,

26.8, 25.9. HRMS (m/z): CioH₈NOBr [M+H]⁺ found: 236.9790. Calculated: 236.9789.

6-Bromo-3.4-dihydro-2//- 1 -benzothiopyran-4-carbonit rile (21c). Ketone 20c (900 mg; 3.70 mmol) and TosMIC (867 mg; 2.63 mmol; 1.1 equiv.) were dissolved in dry THF (10 mL) and cooled to -78 °C. EtOH (259 pL; 4.44 mmol; 1.2 equiv.) and LBuOK (582 mg; 5.18 mmol; 1.4 equiv.) were added sequentially and stirring was continued for 2 h while the cold bath was allowed to warm to room temperature gradually. The cold bath was removed and the mixture was stirred at room temperature for 20 h. Dark red thick solution. The solution was diluted with water, then IN HC1 was added and the mixture was extracted with EtOAc (x2). Organic layers were combined, washed with brine, dried (Na2SO4) and evaporated. Dark red solid. The residue was purified with column chromatography (10% EtOAc in hexanes) to give the product 21c (412 mg, 44%) as white solid.

’H NMR (400 MHz, CDCh) 6 7.48 (d, J = 2.2 Hz, 1H), 7.31 (dd, J = 8.5, 2.2 Hz, 1H), 7.03 (d, J = 8.5 Hz, 1H), 3.99 (dd, J= 6.6, 4.5 Hz, 1H), 3.24 (ddd, = 13.1, 9.0, 4.1 Hz, 1H), 3.09 - 3.02 (m, 1H), 2.50 - 2.33 (m, 2H). ¹³C NMR (101 MHz, CDCh) 6 132.4, 132.3, 132.0, 129.0, 128.8, 119.5, 118.1,

30.9, 26.5, 24.4. HRMS (m/z): CioH₇NSBr [M-H]’ found: 251.9491. Calculated: 251.9483.

Methyl (25)-2-{[(tert-butoxy)carbonyl]amino}-3-(8-cyano-5,6,7,8-tetrahydronaphthalen-2- yl)propanoate (22a). Aryl bromide 21a (1.26 g; 5.32 mmol), Pd2(dba)a (97 mg; 0.11 mmol; 0.02 equiv.) and SPhos (87 mg; 0.21 mmol; 0.04 equiv.) were dissolved in anhydrous DMF (1 mL) under argon atmosphere and freshly prepared solution of alkyl zinc iodide 2 (0.5 M in DMF; 16.0 mL; 7.98 mmol; 1.5 equiv.) was added. The dark red solution was stirred at 65 °C for 3 h. Then the solution was cooled to room temperature and aqueous saturated NH4CI and EtOAc were added. Layers were separated and EtOAc was washed with IN HC1 aqueous solution and brine, dried (Na₇SO4) and evaporated to dryness. Product 22a was purified by column chromatography on silica (30% EtOAc in hexanes) to afford 1.24 g (65%; dr ~1:1) as a orange oil.

’H NMR (400 MHz, CDCh) 5 7.17 - 6.94 (m, 3H), 5.00 (d, J = 8.3 Hz, 1H), 4.66 - 4.46 (m, 1H), 4.00 - 3.88 (m, 1H), 3.76 (s, 1.5H), 3.74 (s, 1.5H), 3.19 - 2.96 (m, 2H), 2.87 - 2.70 (m, 2H), 2.25 - 2.07 (m, 2H), 2.07 - 1.95 (m, 1H), 1.91 - 1.74 (m, 1H), 1.43 (s, 9H). ¹³C NMR (101 MHz, CDCh) 8 172.4, 172.2, 155.2, 135.3, 134.61, 134.55, 130.13, 130.05, 129.7, 129.3, 129.1, 121.83, 121.76, 80.2,

80.1, 54.6, 54.5, 52.6, 52.5, 38.1, 37.8, 30.9, 28.4, 28.3, 28.2, 27.5, 27.4, 21.0, 20.9. HRMS (m/z): C2oH26N₂0₄Na [M+Na]⁺ found: 381.1793. Calculated: 381.1790.

Methyl (2»S)-2-{[(tert-butoxy)carbonyl]amino}-3-(4-cyano-3,4-dihydro-2Z/-l-benzothiopyran- 6-yl)propanoate (22c). Aryl bromide 21c (380 mg; 1.50 mmol), Pd₂(dba)₂ (21 mg; 0.022 mmol; 0.02 equiv.) and SPhos (25 mg; 0.060 mmol; 0.04 equiv.) were dissolved in anhydrous DMF (5 mL) under argon atmosphere and freshly prepared solution of alkyl zinc iodide 2 (0.5 M in DMF; 4.49 mL; 2.24 mmol; 1.5 equiv.) was added. The dark red solution was stirred at 65 °C for 3 h. Then the solution was cooled to room temperature and aqueous saturated NH₄C1 and EtOAc were added. Layers were separated and EtOAc was washed with IN HC1 aqueous solution and brine, dried (Na₂SO₄) and evaporated to dryness. Product 22c was purified by column chromatography on silica (30% EtOAc in hexanes) to afford 500 mg (89%; dr ~1 : 1) as a orange oil.

’H NMR (400 MHz, CDCh) 5 7.11 - 7.04 (m, 2H), 7.01 - 6.93 (m, 1H), 5.00 (d, J = 8.2 Hz, 1H), 4.61 - 4.51 (m, 1H), 4.01 - 3.95 (m, 1H), 3.75 (s, 1.5H), 3.74 (s, 1.5H), 3.28 - 3.16 (m, 1H), 3.14 - 2.95 (m, 3H), 2.46 - 2.34 (m, 2H), 1.43 (s, 9H). ¹³C NMR (101 MHz, CDCh) 8 172.3, 172.1, 155.1, 133.23, 133.18, 131.7, 131.6, 130.6, 130.5, 130.0, 129.9, 127.8, 127.7, 127.1, 127.0, 120.1, 120.0,

80.2, 54.5, 54.4, 52.6, 52.5, 37.9, 37.8, 31.13, 31.11, 28.43, 28.37, 27.0, 26.9, 24.6, 24.5. HRMS (m/z): Ci9H₂₄N₂O₄SNa [M+Na]⁺ found: 399.1365. Calculated: 399.1354.

Methyl 2-{5-bromo-2-[(15)-l-[(25)-2-{[(tert-butoxy)carbonyl]amino}-3-(4-cyano-3,4-dihydro- 2Z/-l-benzopyran-6-yl)propanamido]-2,2-dimethylpropyl]-l,3-oxazol-4-yl}-l,3-oxazole-4- carboxylate (23b). Aryl bromide 21b (480 mg; 2.00 mmol), Pd₂(dba)₂ (36 mg; 0.039 mmol; 0.02 equiv.) and SPhos (33 mg; 0.081 mmol; 0.04 equiv.) were dissolved in anhydrous DMF (3 mL) under argon atmosphere and freshly prepared solution of alkyl zinc iodide 2 (0.5 M in DMF; 6.0 mL; 3.00 mmol; 1.5 equiv.) was added. The dark red solution was stirred at 65 °C for 8 h. Then the solution was cooled to room temperature and aqueous saturated NH₄C1 and EtOAc were added. Layers were separated and EtOAc was washed with IN HC1 aqueous solution and brine, dried (Na₂SO₄) and evaporated to dryness. Negishi coupling product 22b was semi-purified by filtering throug short pad of silica (30% EtOAc in hexanes) to afford 546 mg (75%; dr ~1 : 1) as a colourless oil and used in the next step without further purification.

Then compound 23b was prepared according to General procedure A from crude ester 22b (570 mg; 1.58 mmol), LiOHxJEO (332 mg; 7.91 mmol; 5 equiv.) in THF/water (3: 1 v/v; 6 mL) and amine 4 (567 mg; 1.58 mmol; 1 equiv.), EDC^XHC1 (455 mg; 2.37 mmol; 1.5 equiv.) in pyridine (10 mL) in 1 h. Product 23b was purified by reverse phase flash chromatography (from 10% to 70% MeCN in 0.01% TFA in water) to afford 490 mg (45%) of a 1 : 1 mixture of diastereomers as a white amorphous solid.

‘H NMR (400 MHz, CDCh) 8 8.34 (s, 0.5H), 8.32 (s, 0.5H), 7.14 - 7.06 (m, 1.5H), 7.03 (dd, J= 8.4, 2.2 Hz, 0.5H), 6.74 (d, J= 8.4 Hz, 0.5H), 6.73 - 6.68 (m, 0.5H), 6.68 (d, J= 8.4 Hz, 0.5H), 6.54 (d, J = 7.4 Hz, 0.5H), 5.13 - 4.94 (m, 2H), 4.33 - 4.16 (m, 2.5H), 4.16 - 4.03 (m, 1H), 4.00 - 3.95 (m, 0.5H), 3.94 (s, 3H), 3.05 - 2.92 (m, 2H), 2.32 - 2.18 (m, 2H), 1.43 (s, 9H), 0.96 (s, 4.5H), 0.93 (s, 4.5H). ¹³C NMR (101 MHz, CDCh) 6 170.96, 170.94, 164.9, 164.8, 161.4, 155.6, 154.9, 154.8, 153.1, 153.0, 144.2, 144.0, 134.7, 131.0, 130.7, 130.6, 130.2, 129.5, 129.4, 128.13, 128.10, 123.2, 123.1, 120.6, 120.4, 118.2, 118.0, 115.5, 115.4, 80.6, 63.62, 63.59, 56.1, 55.9, 55.7, 52.4, 37.0, 36.7, 36.0,

35.7, 28.4, 26.81, 26.78, 26.32, 26.25, 26.1. HRMS (m/z): CaiHaeNsOsNaBr [M+Na]⁺ found: 708.1663. Calculated: 708.1645.

Methyl 2-{5-bromo-2-[(15)-l-[(25)-2-{[(tert-butoxy)carbonyl]amino}-3-(8-cyano-5,6,7,8- tetrahydronaphthalen-2-yl)p ropanamido] -2,2-dimethylpropyl] - 1 ,3-oxazol-4-yl}- 1 ,3-oxazole-4- carboxylate (23a). Compound was prepared according to General procedure A from ester 22a (980 mg; 2.73 mmol), LiOHxH2O (574 mg; 13.7 mmol; 5 equiv.) in THF/water (3: 1 v/v; 15 mL) and amine 4 (980 mg; 2.74 mmol; 1 equiv.), EDC^XHC1 (787 mg; 4.10 mmol; 1.5 equiv.) in pyridine (20 mL) in 1 h. Product 23a was purified by reverse phase flash chromatography (from 10% to 70% MeCN in 0.01% TFA in water) to afford 1.44 g (77%) of a 1 : 1 mixture of diastereomers as a white amorphous solid.

’H NMR (400 MHz, CDCh) 6 8.34 (s, 0.5H), 8.32 (s, 0.5H), 7.19 (s, 0.5H), 7.15 (s, 0.5H), 7.10 - 6.93 (m, 2H), 6.81 (d, J= 7.6 Hz, 0.5H), 6.63 (d, J= 7.6 Hz, 0.5H), 5.09 (d, J= 9.3 Hz, 0.5H), 5.09 - 5.02 (m, 0.5H), 5.03 (d, J= 9.3 Hz, 0.5H), 5.06 (dd, J= 23.8, 9.3 Hz, 2H), 5.00 - 4.92 (m, 0.5H), 4.35 - 4.25 (m, 1H), 4.00 - 3.95 (m, 0.5H), 3.94 (s, 3H), 3.93 - 3.88 (m, 0.5H), 3.12 - 2.96 (m, 2H), 2.82 - 2.60 (m, 2H), 2.15 - 2.03 (m, 2H), 2.00 - 1.89 (m, 1H), 1.86 - 1.70 (m, 1H), 1.42 (s, 9H), 0.97 (s, 4.5H), 0.94 (s, 4.5H). ¹³C NMR (101 MHz, CDCh) 8 170.96, 170.95, 164.89, 164.85, 161.4, 155.69, 155.65, 154.83, 154.79, 144.2, 144.0, 135.21, 135.16, 135.1, 134.7, 130.3, 130.2, 130.1,

129.7, 129.1, 128.9, 128.10, 128.07, 123.14, 123.11, 121.9, 121.6, 80.6, 55.9, 55.7, 52.4, 37.2, 36.8, 36.0, 35.7, 30.81, 30.75, 28.4, 28.2, 28.1, 27.4, 26.33, 26.27, 20.81, 20.79. HRMS (m/z): C32H₃8N₅O7NaBr [M+Na]⁺ found: 706.1858. Calculated: 706.1852.

Methyl 2-{5-bromo-2-[(15)-l-[(25)-2-{[(tert-butoxy)carbonyl]amino}-3-(4-cyano-3,4-dihydro- 2Z/-l-benzothiopyran-6-yl)propanamido]-2,2-dimethylpropyl]-l,3-oxazol-4-yl}-l,3-oxazole-4- carboxylate (23c). Compound was prepared according to General procedure A from ester 22c (1 g; 2.66 mmol), LiOHxJBO (557 mg; 13.28 mmol; 5 equiv.) in THF/water (3: 1 v/v; 15 mL) and amine 4 (952 g; 2.66 mmol; 1 equiv.), EDC^XHC1 (764 mg; 3.99 mmol; 1.5 equiv.) in pyridine (15 mL) in 1 h. Product 23c was purified by reverse phase flash chromatography (from 10% to 70% MeCN in 0.01% TFA in water) to afford 1.75 g (94%) of a 1 : 1 mixture of diastereomers as a white amorphous solid.

’H NMR (400 MHz, CDCh) 8 8.34 (s, 0.5H), 8.32 (s, 0.5H), 7.16 (s, 1H), 7.08 - 6.95 (m, 2H), 6.71 (d, J= 8.4 Hz, 0.5H), 6.59 (d, J= 8.4 Hz, 0.5H), 5.11 - 5.00 (m, 1H), 5.05 (d, J= 9.4 Hz, 0.5H), 5.00 (d, J= 9.1 Hz, 0.5H), 5.11 - 5.00 (m, 1H), 4.32 - 4.23 (m, 1H), 4.09 (dd, J= 6.2, 4.3 Hz, 0.5H), 4.03 (dd, J = 6.2, 4.3 Hz, 0.5H), 3.94 (s, 3H), 3.26 - 3.12 (m, 1H), 3.05 - 2.96 (m, 2H), 2.96 - 2.89 (m, 1H), 2.48 - 2.35 (m, 1H), 2.33 - 2.23 (m, 1H), 1.43 (s, 9H), 0.96 (s, 4.5H), 0.94 (s, 4.5H). ¹³C NMR (101 MHz, CDCh) 6 170.84, 170.81, 164.9, 164.8, 161.4, 155.6, 154.9, 154.8, 144.1, 144.0, 134.72, 134.68, 133.6, 133.5, 131.5, 131.4, 131.2, 130.8, 129.8, 129.6, 128.2, 127.8, 127.6, 127.13, 127.06, 123.13, 123.11, 120.1, 119.9, 80.6, 55.9, 55.7, 52.4, 37.2, 37.0, 36.0, 35.8, 30.9, 30.8, 28.4, 26.7, 26.6, 26.30, 26.25, 24.4, 24.3. HRMS (m/z): CaiHaeNsChNaSBr [M+Na]⁺ found: 724.1418. Calculated: 724.1417.

Methyl 2-[(15',65',95)-9-{[(tert-butoxy)carbonyl]amino}-6-tert-butyl-l-cyano-8-oxo-20-oxa-4,7- diazatetracyclo[9.6.2.1², .0¹⁴,¹ ]icosa-2,4,ll,13,18-pentaen-3-yl]-l,3-oxazole-4-carboxylate

(24a). Compound was prepared according to General procedure B from amide 23a (1.34 g; 0.54 mmol) and K3PO4 (2.078 g; 9.79 mmol; 5 equiv.) in DMF (100 mL) in 3 h. Product 24a was purified by reverse phase flash chromatography (from 10% to 70% MeCN in 0.01% TFA in water) to afford 905 mg (77%; dr 96:4) of a single diastereomer as a white amorphous solid.

^XH NMR (400 MHz, CDCh) 6 8.38 (s, 1H), 7.20 (dd, J= 7.9, 1.8 Hz, 1H), 7.13 (d, J= 7.9 Hz, 1H), 6.81 (d, .7= 1.8 Hz, 1H), 5.77 (d, J= 6.4 Hz, 1H), 5.17 (d, J= 9.2 Hz, 1H), 4.63 (d, J= 6.4 Hz, 1H), 3.98 (ddd, J= 12.3, 6.7, 3.1 Hz, 1H), 3.93 (s, 3H), 3.11 (dd, J= 12.1, 12.1 Hz, 1H), 3.08 - 3.00 (m, 1H), 2.95 - 2.86 (m, 1H), 2.86 - 2.76 (m, 2H), 2.28 (ddd, = 13.5, 11.2, 2.4 Hz, 1H), 2.18 - 2.06 (m, 1H), 1.97 - 1.87 (m, 1H), 1.44 (s, 9H), 0.95 (s, 9H). ¹³C NMR (101 MHz, CDCh) 8 171.9, 161.9, 161.4, 155.3, 154.8, 150.5, 144.6, 135.5, 134.8, 134.1, 133.4, 131.9, 130.1, 129.6, 127.5, 119.1, 80.5, 59.0, 57.8, 52.3, 41.1, 38.0, 34.5, 33.4, 28.4, 28.3, 26.6, 20.0. HRMS (m/z): C32H38N5O7 [M+H] found: 604.2778. Calculated: 604.2771. [a]_D ²⁰ -134 (c 1.0, CHCh).

Methyl 2-[(1S,6S,9S)-9-{[(tert-butoxy)carbonyl]amino}-6-tert-butyl-l-cyano-8-oxo-15,20- dioxa-4,7-diazatetracyclo[9.6.2.1², .0¹⁴,¹ ]icosa-2,4,ll,13,18-pentaen-3-yl]-l,3-oxazole-4- carboxylate (24b). Compound was prepared according to General procedure B from amide 23b (360 mg; 0.52 mmol) and K3PO4 (557 mg; 2.62 mmol; 5 equiv.) in DMF (30 mL) in 2 h. Product 24b was purified by reverse phase flash chromatography (from 10% to 70% MeCN in 0.01% TFA in water) to afford 251 mg (79%; dr 99: 1) of a single diastereomer as a white amorphous solid.

¹H NMR (400 MHz, CDCh) δ 8.37 (s, 1H), 7.19 (dd, J= 8.4, 2.2 Hz, 1H), 6.87 (d, J= 8.4 Hz, 1H), 6.81 (d, .7= 2.2 Hz, 1H), 5.91 (d, J = 6.3 Hz, 1H), 5.16 (d, J= 9.2 Hz, 1H), 4.65 (d, J= 6.3 Hz, 1H), 4.48 - 4.30 (m, 2H), 4.00 (ddd, J = 12.2, 9.2, 3.2 Hz, 1H), 3.93 (s, 3H), 3.26 (d, J = 14.3 Hz, 1H), 3.13 (dd, J = 12.1, 12.1 Hz, 1H), 2.74 (dd, J = 12.5, 3.2 Hz, 1H), 2.39 (ddd, J = 14.3, 10.4, 4.7 Hz, 1H), 1.44 (s, 9H), 0.97 (s, 9H). ¹³C NMR (101 MHz, CDCh) δ 172.1, 162.0, 161.3, 155.4, 154.6, 153.6, 148.6, 144.7, 134.7, 133.7, 131.6, 128.6, 128.1, 118.2, 118.1, 117.3, 80.6, 63.0 59.0, 57.7, 52.4, 37.4, 37.3, 33.52, 33.49, 28.4, 26.7. HRMS (m/z): C31H35N5O8Na [M+Na]⁺ found: 628.2388. Calculated: 628.2383. [a]_D ²⁰ -151 (c 1.0, CHCh).

Methyl 2-[(1S,6S,9S)-9-{[(tert-butoxy)carbonyl]amino}-6-tert-butyl-l-cyano-8-oxo-20-oxa-15- thia-4,7-diazatetracyclo[9.6.2.1², .0¹⁴,¹ ]icosa-2,4,ll,13,18-pentaen-3-yl]-l,3-oxazole-4- carboxylate (24c). Compound was prepared according to General procedure B from amide 23c (1.20 g; 1.71 mmol) and K3PO4 (1.81 g; 8.54 mmol; 5 equiv.) in DMF (80 mL) in 2 h. Product 24c was purified by reverse phase flash chromatography (from 10% to 70% MeCN in 0.01% TFA in water) to afford 705 mg (66%; dr 99: 1) of a single diastereomer as a white amorphous solid.

’H NMR (400 MHz, CDCh) δ 8.37 (s, 1H), 7.17 - 7.09 (m, 2H), 6.79 (d, J = 1.6 Hz, 1H), 5.92 (d, J = 6.1 Hz, 1H), 5.15 (d, J = 9.2 Hz, 1H), 4.64 (d, J= 6.0 Hz, 1H), 4.00 (ddd, J = 12.1, 9.1, 3.1 Hz, 1H), 3.93 (s, 3H), 3.53 (ddd, J = 13.9, 5.8, 1.9 Hz, 1H), 3.46 (ddd, J = 13.9, 12.1, 1.9 Hz, 1H), 3.09 (dd, J= 12.1, 12.1 Hz, 1H), 3.01 (ddd, J = 13.9, 5.8, 1.9 Hz, 1H), 2.74 (dd, J = 12.4, 3.1 Hz, 1H), 2.46 (ddd, J = 13.9, 12.1, 2.3 Hz, 1H), 1.44 (s, 9H), 0.97 (s, 9H). ¹³C NMR (101 MHz, CDCh) δ 172.0, 162.3, 161.3, 155.3, 154.5, 149.3, 144.7, 134.8, 134.7, 132.4, 132.1, 130.1, 128.0, 127.8, 127.8, 117.9, 80.6, 59.2, 57.5, 52.3, 40.7, 37.7, 34.5, 33.4, 28.4, 26.7, 23.6. HRMS (m/z): C31H35N5O7NaS [M+Na]⁺ found: 644.2174. Calculated: 644.2155. [a]_D ²⁰ -82 (c 1.0, CHCh).

Methyl 2-[(1S,6S,9S)-6-tert-butyl-l-cyano-9-[(2S)-2-hydroxy-3-methylbutanamido]-8-oxo-20- oxa-4,7-diazatetracyclo[9.6.2.1², .0¹⁴,¹ ]icosa-2,4,ll,13,18-pentaen-3-yl]-l,3-oxazole-4- carboxylate (DZA-141). Compound was prepared according to General procedure C from macrocycle 24a (135 mg; 0.22 mmol), TFA (172 pL; 2.24 mmol; 10 equiv.) in DCM (1 mL) in 1 h and 5-HzVA (29 mg; 0.25 mmol; 1.5 equiv.), EDOHCl (63 mg; 0.33 mmol; 2 equiv.), HOBt (67 mg; 0.50 mmol; 3 equiv.), DIPEA (143 pL; 0.82 mmol; 5 equiv.) in DMF (2 mL) in 1 h . Product DZA-141 was purified by reverse phase flash chromatography (10% to 50% MeCN in 0.01% TFA in water) to afford 68 mg (68%) as a white amorphous solid.

^XH NMR (400 MHz, MeOD) 5 8.76 (s, 1H), 8.43 (d, J = 6.1 Hz, 1H), 7.31 (dd, J = 7.9, 1.8 Hz, 1H), 7.22 (d, .7= 7.9 Hz, 1H), 6.69 (d, J= 1.8 Hz, 1H), 4.64 (dd, J= 11.8, 3.7 Hz, 1H), 4.54 (d, = 6.1 Hz, 1H), 3.94 (s, 3H), 3.86 (d, J = .1 Hz, 1H), 3.11 - 3.00 (m, 2H), 2.97 - 2.84 (m, 2H), 2.82 (d, J = 12.4, 3.7 Hz, 1H), 2.31 (ddd, J= 13.7, 11.0, 2.8 Hz, 1H), 2.15 - 2.03 (m, 2H), 2.03 - 1.94 (m, 1H), 1.00 (d, J= 6.9 Hz, 3H), 0.98 - 0.94 (m, 9H), 0.89 (d, J= 6.9 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.9, 174.1, 164.4, 162.8, 156.2, 152.2, 146.8, 137.0, 135.5, 135.3, 134.0, 133.2, 131.2, 131.1, 128.0, 120.5, 76.8, 61.1, 55.9, 52.7, 42.4, 39.2, 35.3, 34.2, 33.2, 29.0, 26.7, 21.0, 19.5, 16.3. HRMS (m/z): C32H38N5O7 [M+H]⁺ found: 604.2783. Calculated: 604.2771. [a]_D ²⁰ -165 (c 1.0, MeOH).

Methyl 2-[(15',65',95)-6-tert-butyl-l-cyano-9-[(25)-2-hydroxy-3-methylbutanamido]-8-oxo- 15,20-dioxa-4,7-diazatetracyclo[9.6.2.1², .0¹⁴,¹ ]icosa-2,4,ll,13,18-pentaen-3-yl]-l,3-oxazole-4- carboxylate (DZA-140). Compound was prepared according to General procedure C from macrocycle 24b (100 mg; 0.17 mmol), TFA (127 pL; 1.65 mmol; 10 equiv.) in DCM (1 mL) in 1 h and 5-HzVA (29 mg; 0.25 mmol; 1.5 equiv.), EDC^XHC1 (63 mg; 0.33 mmol; 2 equiv.), HOBt (67 mg; 0.49 mmol; 3 equiv.), DIPEA (142 pL; 0.82 mmol; 5 equiv.) in DMF (1 mL) in 1 h . Product DZA-140 was purified by reverse phase flash chromatography (10% to 50% MeCN in 0.01% TFA in water) to afford 40 mg (40%) as a white amorphous solid.

‘HNMR (400 MHz, MeOD) 5 8.76 (s, 1H), 7.31 (dd, J= 8.5, 2.1 Hz, 1H), 6.92 (d, J= 8.5 Hz, 1H), 6.67 (d, J= 2.1 Hz, 1H), 4.65 (dd, J = 11.8, 3.7 Hz, 1H), 4.57 (s, 1H), 4.46 (ddd, J = 11.8, 3.3, 3.3 Hz, 1H), 4.32 (ddd, J= 11.8, 11.8, 1.6 Hz, 1H), 3.94 (s, 3H), 3.86 (d, J= 3.8 Hz, 1H), 3.22 (ddd, J= 14.6, 3.3, 1.6 Hz, 1H), 3.06 (dd, J= 12.2, 12.2 Hz, 1H), 2.77 (dd, J= 12.6, 3.8 Hz, 1H), 2.42 (ddd, J = 14.6, 11.8, 3.3 Hz, 1H), 2.09 (sept d, J= 6.9, 3.8 Hz, 1H), 1.00 (d, J= 6.9 Hz, 3H), 0.99 (s, 9H), 0.88 (d, J= 6.9 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.9, 174.2, 164.4, 162.8, 155.9, 155.0, 150.5, 146.9, 135.5, 134.2, 133.1, 129.8, 128.6, 119.5, 119.2, 118.8, 76.8, 63.9, 60.9, 55.8, 52.7, 38.8, 38.6, 34.3, 34.2, 33.2, 26.8, 19.5, 16.3. HRMS (m/z): C31H36N5O8 [M+H]⁺ found: 606.2565. Calculated: 606.2564. [a]_D ²⁰ -194 (c 1.0, MeOH).

Methyl 2-[(15',65',95)-6-tert-butyl-l-cyano-9-[(25)-2-hydroxy-3-methylbutanamido]-8-oxo-20- oxa-15-thia-4,7-diazatetracyclo[9.6.2.1², .0¹⁴,¹ ]icosa-2,4,ll,13,18-pentaen-3-yl]-l,3-oxazole-4- carboxylate (DZA-144). Compound was prepared according to General procedure C from macrocycle 24c (200 mg; 0.32 mmol), TFA (247 pL; 3.22 mmol; 10 equiv.) in DCM (2 mL) in 1 h and 5-HzVA (57 mg; 0.48 mmol; 1.5 equiv.), EDOHCl (124 mg; 0.64 mmol; 2 equiv.), HOBt (131 mg; 0.97 mmol; 3 equiv.), DIPEA (279 pL; 1.61 mmol; 5 equiv.) in DMF (2 mL) in 1 h . Product DZA-144 was purified by reverse phase flash chromatography (10% to 50% MeCN in 0.01% TFA in water) to afford 120 mg (60%) as a white amorphous solid.

‘HNMR (400 MHz, MeOD) 5 8.76 (s, 1H), 8.57 (d, J= 6.0 Hz, 1H), 7.27 (dd, J= 8.2, 1.8 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 6.68 (d, J = 1.8 Hz, 1H), 4.65 (dd, J = 11.8, 3.7 Hz, 1H), 4.60 - 4.55 (m, 1H), 3.94 (s, 3H), 3.86 (d, 3.7 Hz, 1H), 3.50 (ddd, J= 14.2, 5.7, 1.8 Hz, 1H), 3.40 (ddd, J= 13.7,

12.0, 1.8 Hz, 1H), 3.12 (ddd, J= 13.7, 5.7, 2.3 Hz, 1H), 3.04 (dd, J= 12.1, 12.1 Hz, 1H), 2.77 (dd, J = 12.5, 3.7 Hz, 1H), 2.48 (ddd, J= 14.2, 12.0, 2.3 Hz, 1H), 2.09 (sept d, J = 6.9, 3.7 Hz, 1H), 1.00 (d, J= 6.9 Hz, 3H), 0.99 (s, 9H), 0.88 (d, = 6.9 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.9, 174.1, 164.6, 162.8, 155.9, 151.1, 146.9, 135.5, 135.4, 133.53, 133.49, 131.6, 129.4, 128.7, 128.4, 119.2, 76.8, 61.1, 55.6, 52.7, 42.0, 39.0, 35.3, 34.2, 33.1, 26.8, 24.2, 19.5, 16.3. HRMS (m/z): C31H36N5O7S [M+H]⁺ found: 622.2345. Calculated: 622.2335. [a]_D ²⁰ -129 (c 1.0, MeOH).

Methyl 2- [( 15,65,95)-6-tert-butyl- l-cyano-9- [(25)-2-hydroxy-3-methylbutanamido] -8,15,15- trioxo-20-oxa-15k -thia-4,7-diazatetracyclo[9.6.2.1², .0¹⁴,¹ ]icosa-2,4,ll,13,18-pentaen-3-yl]- l,3-oxazole-4-carboxylate (DZA-145). To a sulfide DZA-144 (45 mg; 0.072 mmol) in dry DCM (0.5 mL) was added m-chloroperoxybenzoic acid (73%; 51 mg; 0.22 mmol; 3 equiv.) and stirred for 30 minutes at room temperature. The reaction mixture was diluted with DCM and washed with saturated NaHCO3 and brine. The organic layer was dried (Na2SO4) and evaporated. White amorphous solid was purified with reverse phase flash chromatography (10% to 50% MeCN in 0.01% TFA in water) to give sulfone DZA-145 (45 mg; 95%) as a white amorphous solid.

'H NMR (400 MHz, MeOD) 5 8.78 (s, 1H), 8.51 (d, J = 5.8 Hz, 1H), 7.98 (d, J = 8.2 Hz, 1H), 7.75 (dd, J = 8.2, 1.6 Hz, 1H), 7.02 (d, J = 1.6 Hz, 1H), 4.72 (dd, J = 11.8, 3.9 Hz, 1H), 4.55 - 4.52 (m, 1H), 3.95 (s, 3H), 3.87 (d, J= .1 Hz, 1H), 3.82 - 3.65 (m, 2H), 3.59 - 3.51 (m, 1H), 3.40 - 3.32 (m, 1H), 3.17 (dd, J = 12.0, 12.0 Hz, 1H), 3.00 (dd, J= 12.2, 3.9 Hz, 1H), 2.09 (sept d, J = 6.9, 3.9 Hz, 1H), 1.00 (d, J= 6.9 Hz, 3H), 0.98 (s, 9H), 0.89 (d, J= 6.9 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.9, 173.5, 164.8, 162.8, 155.7, 148.5, 147.0, 142.9, 138.8, 135.6, 135.0, 133.4, 133.0, 129.2, 125.3, 118.0, 76.9, 61.2, 55.4, 52.8, 47.8, 42.1, 39.2, 34.3, 33.2, 32.0, 26.7, 19.5, 16.3. HRMS (m/z): C31H36N5O9S [M+H]⁺ found: 654.2247. Calculated: 654.2234. [a]_D ²⁰ -130 (c 1.0, MeOH).

The synthesis of diazonamide A analogs DZA-147, 154-155 is shown in Scheme 6.

Modifications in the quaterinary position of macrocycle are depicted in Scheme 6. Accordingly, amide 25 was obtained from corresponding macrocycle 6 after conversion of nitrile to amide in the presence of K2CO3 and hydrogen peroxide. The resulting amide 25 was converted into analog DZA- 147 after A-Boc cleavage and following amide coupling with carboxylic acid 7. Primary amine containing analog DZA-154 was obtained from analog DZA-129 after reduction of nitrile in the presence of InCh and NaBHj. Methyl ester containing analog DZA-155 was synthesized from analog DZA-129 in two steps. In the first step DZA-129 was treated with LiOHxH2O in methanol/water mixture to produce methyl ester at the quaterinary position. In the second step methyl ester at the oxazole moiety was restored with TMSCHN2 after it was hydrolyzed during the first step (Scheme 6).

Scheme 6. Synthesis of diazonamide A analogs DZA-147 and DZA-154,

Methyl 2-[(l l?,65,9»S)-9-{[(tert-butoxy)carbonyl]amino}-6-tert-butyl-l-carbamoyl-8-oxo-19- oxa-4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-3-yl]-l,3-oxazole-4- carboxylate (25). To nitrile 6 (115 mg; 0.20 mmol) in dry DMSO (0.5 mL) K2CO3 (54 mg; 0.39 mmol; 2 equiv.) was added and the flask was cooled to 0°C. Then H2O2 (50% in water; 117 pL; 1.95 mmol; 10 equiv.) was added dropwise and the resulting suspension was warmed to room temperature and stirred for 3 h. Then the suspension was quenched by the addition of aqueous IN Na2S20a and extracted with EtOAc (x2). Organic layer was washed with brine, dried (Na2SO4) and evaporated. The resulting yellow oil was purified with reverse phase flash chromatography (from 10% to 50% MeCN in 0.01% TFA in water) to give amide 25 (58 mg; 49%) as a yellowish solid.

’H NMR (400 MHz, CDCh) 8 8.31 (s, 1H), 7.47 (s, 1H), 7.16 - 7.09 (m, 2H), 5.69 (d, J= 9.1 Hz, 1H), 5.27 (d, J= 9.2 Hz, 1H), 4.97 (d, J= 9.2 Hz, 1H), 3.93 (s, 3H), 3.87 (ddd, J= 12.3, 9.2, 3.3 Hz, 1H), 3.30 (dd, J= 12.1, 12.1 Hz, 1H), 3.16 - 3.06 (m, 1H), 3.04 - 2.80 (m, 3H), 2.79 - 2.69 (m, 1H), 1.72 (s, 2H), 1.43 (s, 9H), 1.09 (s, 9H). ¹³C NMR (101 MHz, CDCh) 6 172.6, 172.3, 161.3, 160.8, 156.4, 155.3, 153.2, 144.2, 142.5, 142.2, 134.3, 134.2, 131.6, 129.3, 125.2, 125.0, 80.4, 60.9, 58.7, 56.7, 52.4, 38.4, 34.7, 33.4, 31.3, 28.4, 26.6. HRMS (m/z): C31H38N5O8 [M+H]⁺ found: 608.2712. Calculated: 608.2720. [a]_D ²⁰ - 55 (c 1.0, CHCh). Methyl 2-|( 1 /?.6.S.9.S')-6-/‘ r/‘-biityl-l-c:irb:inioyl-9-|(2.S)-2-hydroxy-3-niethylbiitanainido|-8- oxo-19-oxa-4,7-diazatetracyclo[9.5.2.1², .O¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-3-yl]-l,3- oxazole-4-carboxylate (DZA-147). Compound was prepared according to General procedure C from macrocycle 25 (50 mg; 0.082 mmol), TFA (63 pL; 82 mmol; 10 equiv.) in DCM (1 mL) in 1 h and N-H/VA (15 mg; 0.12 mmol; 1.5 equiv.), EDC^XHC1 (32 mg; 0.17 mmol; 2 equiv.), HOBt (34 mg; 0.25 mmol; 3 equiv.), DIPEA (72 pL; 0.41 mmol; 5 equiv.) in DMF (1 mL) in 1 h. Product DZA-147 was purified by reverse phase flash chromatography (10% to 50% MeCN in 0.01% TFA in water) to afford 32 mg (64%) as a white amorphous solid.

’H NMR (400 MHz, MeOD) 5 8.64 (s, 1H), 7.29 - 7.13 (m, 3H), 4.87 - 4.82 (m, 1H), 4.52 (dd, J = 11.8, 3.6 Hz, 1H), 3.92 (s, 3H), 3.86 (d, J= 3.8 Hz, 1H), 3.26 - 3.13 (m, 3H), 2.84 (dd, J= 12.4, 3.7 Hz, 1H), 2.82 - 2.67 (m, 2H), 2.09 (sept d, J= 6.9, 3.8 Hz, 1H), 1.06 (s, 9H), 1.01 (d, J= 6.9 Hz, 3H), 0.90 (d, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.7, 175.5, 174.3, 162.8, 162.7, 157.7, 155.3, 146.2, 145.2, 143.6, 135.5, 135.1, 130.8, 130.6, 126.6, 125.9, 76.8, 62.2, 58.9, 56.6, 52.6, 39.6, 36.4, 34.4, 33.2, 32.5, 26.8, 19.5, 16.3. HRMS (m/z): C31H38N5O8 [M+H]⁺ found: 608.2711. Calculated: 608.2720. [a]_D ²⁰ -60 (c 1.0, MeOH).

Methyl 2-[(lS,6S,9S)-l-(aminomethyl)-6-tert-butyl-9-[(2S)-2-hydroxy-3-methylbutanamido]- 8-oxo-19-oxa-4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-3-yl]-l,3- oxazole-4-carboxylate (DZA-154). Anhydrous InCF (30 mg; 0.14 mmol, 1 equiv.) and NaBHj (15 mg; 0.41 mmol; 3 equiv.) were suspended in dry THF (1) at room temperature under argon atmosphere. The green suspension was stirred for 1 h at room temperature, then macrocycle DZA- 129 (80 mg; 0. 14 mmol; 1 equiv.) was added in one portion. The resulting suspension was stirred for another 2 h at the same temperature. Then the reaction mixture was quenched by the addition of aqueous IN HC1, then NH4OH was added dropwise until the mixture reached basic pH. The resulting mixture was extracted with EtOAc, organic layer was washed witn brine, dried (Na2SC>4) and evaporated. The residue was purified with reverse phase flash chromatography (from 10% to 40% MeCN in aqueous 0,1% TFA) to give amine DZA-154 (40 mg; 50%) as a white amorphous solid.

’H NMR (400 MHz, MeOD) 5 8.73 (s, 1H), 8.44 (d, J= 8.2 Hz, 1H), 7.31 - 7.23 (m, 2H), 7.04 (d, J = 1.4 Hz, 1H), 4.89 - 4.82 (m, 1H), 4.48 (dd, J= 11.7, 3.7 Hz, 1H), 4.09 (d, J= 13.3 Hz, 1H), 3.96 (s, 3H), 3.91 (d, J= 13.3 Hz, 1H), 3.87 (d, = 3.7 Hz, 1H), 3.25 - 3.08 (m, 2H), 2.91 - 2.81 (m, 2H), 2.81 - 2.72 (m, 1H), 2.58 - 2.47 (m, 1H), 2.09 (sept d, J= 6.8, 3.7 Hz, 1H), 1.06 (s, 9H), 1.01 (d, J= 6.8 Hz, 3H), 0.89 (d, J = 6.8 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.8, 174.2, 163.8, 163.2, 157.5, 154.2, 146.6, 144.8, 143.5, 136.0, 134.9, 131.2, 129.9, 127.0, 126.6, 76.8, 58.8, 56.5, 54.5, 52.9, 45.0, 39.5, 36.0, 34.2, 33.2, 31.3, 26.7, 19.5, 16.3. HRMS (m/z): C31H40N5O7 [M+H]⁺ found: 594.2935. Calculated: 594.2928. [a]_D ²⁰ -142 (c 1.0, MeOH).

The synthesis of diazonamide A analogs DZA-137 and DZA-142 is shown in Scheme 7.

Accordingly, analog DZA-137 was prepared in three steps from macrocycle 6. Methyl ester was reduced with LiBHj in TFE/THF and then after A-Boc cleavage, crude amine was coupled with fS')- 2-hydroxy-3 -methylbutanoic acid fS'-H/VA; 7) to give analog DZA-137. Nitrile moiety containing analog DZA-142 was prepared from ester 6. Methyl ester first was converted into amide in the presence of NH3 and then resulting amide was treated with TFAA and pyridine to give nitrile 27. NBoc cleavage and following amide coupling with 5-HzVA 7 gave analog DZA-142 (Scheme 7).

Scheme 7. Synthesis of diazonamide A analogs DZA-137 and DZA-142, tert- Butyl V-[( l.S.6.S.9.S)-6-/ /7-butyl-l-cyano-3-|4-(hydroxy methyl)- 1 ,3-oxazol-2-yl]-8-oxo-l 9- oxa-4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-9-yl]carbamate (26). To a solution of ester 6 (120 mg; 0.20 mmol) in dry THF (1 mL) TFE (146 pL, 2.04 mmol, 10 equiv.) was added and flask was cooled to 0°C. Then LiBTU (22 mg; 1.02 mmol; 5 equiv.) was added in one portion and resulting suspension was stirred for 1 h at 0°C, then gradually warmed to room temperature and stirred for 2 h. The reaction mixture was quenched by the addition of aqueous IN HC1 and extracted with EtOAc (x2). Organic layers were combined, dried (Na2SO4), evaporated. The off-white amorphous solid was purified with reverse flash column chromatography (10% to 70% MeCN in 0.01% TFA in water) to give alcohol 26 (90 mg; 79%) as a white amorphous solid.

’H NMR (400 MHz, MeOD) 5 7.99 (t, J= 1.1 Hz, 1H), 7.35 - 7.27 (m, 1H), 7.01 (s, 1H), 4.75 (s, 1H), 4.63 (d, J = 1.1 Hz, 2H), 4.13 (dd, J= 11.9, 3.6 Hz, 1H), 3.28 - 3.21 (m, 1H), 3.18 - 3.04 (m, 2H), 2.99 - 2.89 (m, 2H), 2.85 (dd, J= 12.4, 3.6 Hz, 1H), 1.44 (s, 9H), 1.03 (s, 9H). ¹³C NMR (101 MHz, MeOD) 5 174.8, 164.2, 157.2, 155.7, 149.3, 144.0, 142.6, 142.4, 138.0, 137.2, 131.8, 129.7, 128.4, 126.5, 119.6, 80.6, 59.5, 58.1, 57.3, 47.7, 39.2, 38.8, 34.3, 31.7, 28.7, 26.8. HRMS (m/z): C30H36N5O6 [M+H]⁺ found: 562.2677. Calculated: 562.2666. [a]_D ²⁰ -139 (c 1.0, MeOH).

(2S)-N- 1 ( 15.6.S'.9.S')-6-/crt- Buty I- l-cyano-3- [4-(hydroxy methyl)- 1 ,3-oxazol-2-yl] -8-oxo- 19-oxa- 4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-9-yl]-2-hydroxy-3- methylbutanamide (DZA-137). Compound was prepared according to General procedure C from macrocycle 26 (90 mg; 0. 16 mmol), TFA (123 pL; 1.60 mmol; 10 equiv.) in DCM (1 mL) in 1 h and 5-HzVA (29 mg; 0.24 mmol; 1.5 equiv.), EDC^XHC1 (62 mg; 0.32 mmol; 2 equiv.), HOBt (65 mg; 0.48 mmol; 3 equiv.), DIPEA (139 pL; 0.80 mmol; 5 equiv.) in DMF (2 mL) in 1 h. Product DZA- 137 was purified by reverse phase flash chromatography (10% to 50% MeCN in 0.01% TFA in water) to afford 25 mg (28%) as a white amorphous solid.

’H NMR (400 MHz, MeOD) 5 7.99 (t, J= 1.1 Hz, 1H), 7.41 - 7.27 (m, 2H), 6.99 (s, 1H), 4.73 (s, 1H), 4.63 (d, J = 1.1 Hz, 2H), 4.55 (dd, J = 11.8, 3.8 Hz, 1H), 3.86 (d, J = 3.8 Hz, 1H), 3.27 - 3.04 (m, 3H), 3.01 - 2.81 (m, 3H), 2.09 (sept d, J = 6.9, 3.8 Hz, 1H), 1.01 (s, 9H), 1.00 (d, = 6.9 Hz, 3H), 0.89 (d, J= 6.9 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.8, 173.9, 164.1, 155.7, 149.4, 144.0, 142.8, 142.6, 138.1, 136.8, 131.8, 129.7, 128.5, 126.6, 119.6, 76.8, 59.6, 57.3, 56.3, 47.7, 39.3, 39.2, 34.3, 33.2, 31.7, 26.8, 19.5, 16.3. HRMS (m/z): C30H36N5O6 [M+H]⁺ found: 562.2675. Calculated: 562.2666. [a]_D ²⁰ -142 (c 1.0, MeOH).

ZczZ- Butyl A-[(15',65',95)-6-tert-butyl-l-cyano-3-(4-cyano-l,3-oxazol-2-yl)-8-oxo-19-oxa-4,7- diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-9-yl]carbamate (27). To a solution of ester 6 (250 mg; 0.42 mmol) in dry MeOH (2 mL) NH3 (7M in MeOH; 1.82 mL; 12.72 mmol, 30 equiv.) was added dropwise at room temperature. The yellowish solution was stirred for 18 h, then evaporated. Crude amide (220 mg; 90%) was used in the next step without additional purification. To a crude amide (150 mg; 0.26 mmol) in dry dioxane (1 mL) was added pyridine (106 pL; 1.31 mmol, 5 equiv.) and the resulting solution was cooled to 0°C. Then TFAA (109 pL; 0.78 mmol; 3 equiv.) was added dropwise and the flask was warmed to room temperature gradually. Solution became red after 1 h of stirring. Then the red solution was quenched by the addition of aqueous saturated NH4CI and extracted with EtOAc (x2). Organic layers were combined, washed with brine, dried (Na2SO4), evaporated. The resulting red amorphous material was purified with reverse phase column chromatography (10% to 70% MeCN in 0.01% TFA in water) to give nitrile 27 (75 mg; 52%) as a white amorphous solid.

^XH NMR (400 MHz, CDCh) 8 8.31 (s, 1H), 7.26 (s, 2H), 7.11 (s, 1H), 5.82 (d, J= 7.5 Hz, 1H), 5.26 (d, J = 9.2 Hz, 1H), 4.76 (d, J= 7.5 Hz, 1H), 3.92 (ddd, J = 12.3, 9.2, 3.4 Hz, 1H), 3.35 - 3.12 (m, 3H), 3.01 - 2.91 (m, 1H), 2.91 - 2.77 (m, 2H), 1.43 (s, 9H), 1.00 (s, 9H). ¹³C NMR (101 MHz, CDC1₃) 5 171.9, 162.1, 155.6, 155.4, 149.8, 146.6, 141.4, 140.4, 135.6, 130.4, 129.5, 126.4, 125.6, 118.0, 116.7, 111.5, 80.6, 58.2, 58.1, 46.4, 38.5, 37.8, 33.6, 31.1, 28.4, 26.6. HRMS (m/z): Cao^NeOsNa [M+Na]⁺ found: 579.2327. Calculated: 579.2332. [a]_D ²⁰ -152 (c 1.0, CHCh).

(2S)-N- [(15',65',95)-6-terCButyl- l-cyano-3-(4-cyano- 1 ,3-oxazol-2-yl)-8-oxo- 19-oxa-4,7 - diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-9-yl]-2-hydroxy-3- methylbutanamide (DZA-142). Compound was prepared according to General procedure C from macrocycle 27 (70 mg; 0.13 mmol), TFA (97 pL; 1.26 mmol; 10 equiv.) in DCM (0.5 mL) in 1 h and 5-HzVA (19 mg; 0.16 mmol; 1.5 equiv.), EDC^XHC1 (41 mg; 0.22 mmol; 2 equiv.), HOBt (44 mg; 0.32 mmol; 3 equiv.), DIPEA (93 pL; 0.54 mmol; 5 equiv.) in DMF (1 mL) in 1 h. Product DZA-142 was purified by reverse phase flash chromatography (10% to 50% MeCN in 0.01% TFA in water) to afford 16 mg (27%) as a white amorphous solid.

‘H NMR (400 MHz, MeOD) 5 8.90 (s, 1H), 7.42 - 7.26 (m, 2H), 6.98 (s, 1H), 4.71 (s, 1H), 4.56 (dd, J= 11.8, 3.8 Hz, 1H), 3.86 (d, J= 3.8 Hz, 1H), 3.35 - 3.26 (m, 1H), 3.23 - 3.10 (m, 2H), 3.04 - 2.95 (m, 1H), 2.94 - 2.86 (m, 2H), 2.08 (sept d, J = 6.9, 3.8 Hz, 1H), 1.00 (s, 9H), 1.00 (d, = 6.8 Hz, 3H), 0.90 (d, J= 6.8 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.8, 173.9, 164.3, 156.8, 151.3, 149.8, 142.9, 142.1, 136.8, 131.9, 129.8, 127.2, 126.6, 119.3, 117.0, 112.5, 76.8, 59.8, 56.3, 47.8, 39.3, 39.1, 34.4, 33.2, 31.8, 26.7, 19.5, 16.3. HRMS (m/z): C30H33N6O5 [M+H]⁺ found: 557.2521. Calculated: 557.2512. [a]_D ²⁰ -153 (c 1.0, MeOH).

The synthesis of diazonamide A analogs DZA-166, DZA-174 and DZA-175 is shown in Scheme 8.

The synthesis of fluorinated analogs is depicted in Scheme 8. Mono-fluorinated analog DZA-166 was obtained from corresponding alcohol 26. Alcohol was treated with MsCl under basic conditions to give mesylate, which was subjected to SN2-type reaction in the presence of TBAF. Fluorinated macrocycle 28 was then subjected into A-Boc deprotection followed by amide bond formation with 5-HzVA 7 to give analog DZA-166. Di-fluorinated analog DZA-174 was obtained from alcohol 26. Alcohol was oxidized to aldehyde with DMP and then treated with DAST reagent to furnish difluoride 29. After A-Boc cleavage with TFA the resulting amine was coupled with (5)-2-hydroxy-3- methylbutanoic acid fS’-HzVA; 7) to give analog DZA-174. Tri-fluorinated analog DZA-175 was obtained starting from ester 3. Ester 3 was hydrolyzed with LiOHxH2O to produce carboxylic acid, which was coupled with corresponding amine 30 to give amide 31. Amide 31 was then subjected into macrocyclization under basic conditions to give macrocycle 32 with diastereomeric ratio 99: 1. Methyl ester of 32 was then hydrolyzed and coupled with 2-amino-3,3,3-trifluoropropan-l-ol to give amide 33. Primary alcohol of 33 was oxidized to aldehyde and after futher oxazole cyclization reaction in the presence of PPh3 and 1,2-dibromotetrachloroethane under basic conditions was converted into trifluorinated bioxazole 34. After A-Boc cleavage with TFA the resulting amine was coupled with (S)- 2-hydroxy-3 -methylbutanoic acid (S-HiVA; 7) to give analog DZA-175 (Scheme 8).

Scheme 8. Synthesis of diazonamide A analogs DZA-166, DZA-174 and DZA-175, tert- Butyl \-|( l.S.6.S.9.S)-6-tert-biityl-l-cyano-3-|4-(fluoromethyl)-l .3-oxazol-2-yl|-8-oxo-l 9- oxa-4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-9-yl]carbamate (28). To alcohol 26 (178 mg; 0.32 mmol) in dry THF (1 mL) TEA (88 qL; 0.63 mmol; 2 equiv.) was added followed by DMAP (1 mg; 0.01 mmol; 0.03 equiv.). Then flask was cooled to 0°C and MsCl (37 qL; 0.48 mmol; 1.5 equiv.) was added dropwise. The orange solution was warmed to room temperature and stirred for 1 h. Then the solution was diluted with EtOAc and washed with IN HC1 (x2). The organic layer was washed with brine, dried (Na2SO4) and evaporated. The resulting yellow oil was used in the next step without additional purification. To a crude mesylate (203 mg; 0.32 mmol) in dry THF (1 mL) TBAF (IM in THF; 3.17 mL; 3.17 mmol; 10 equiv.) was added dropwise at room temperature. The resulting yellow solution was stirred for 2 h. Then the solution was diluted with EtOAc and washed with aqueous saturated NH4CI (x2). Organic layers were washed with brine, dried (Na2SO4) and evaporated. The yellow oil was purified with reverse phase flash chromatograhy (10% to 70% MeCN in 0.01% TFA in water) to give fluoride 28 (135 mg; 76%) as a white amorphous solid.

‘HNMR (400 MHz, CDCh) 8 7.88 (d, J= 4.3 Hz, 1H), 7.25 - 7.20 (m, 2H), 7.11 (s, 1H), 5.78 (d, J = 7.8 Hz, 1H), 5.51 - 5.43 (m, 1H), 5.39 - 5.32 (m, 1H), 5.27 (d, J= 9.2 Hz, 1H), 4.77 (d, = 7.8 Hz, 1H), 3.92 (ddd, J= 12.4, 9.2, 3.4 Hz, 1H), 3.33 - 3.21 (m, 2H), 3.21 - 3.12 (m, 1H), 3.01 - 2.90 (m, 1H), 2.90 - 2.79 (m, 2H), 1.43 (s, 9H), 1.00 (s, 9H). ¹³C NMR (101 MHz, CDCh) 6 171.9, 161.8,

155.3, 154.9, 148.1, 141.4, 140.9, 138.1, 138.0 (d, J= 19.7 Hz), 135.5, 130.2, 129.5, 127.7, 125.5,

118.3, 80.5, 76.4 (d, J= 165.2 Hz), 58.3, 58.0, 46.4, 38.7, 37.9, 33.5, 31.1, 28.4, 26.6. HRMS (m/z): C₃oH34N50₅FNa [M+Na]⁺ found: 586.2439. Calculated: 586.2442. [a]_D ²⁰ -126 (c 1.0, MeCN).

(25)- N- 1 ( l .S.6.S.9.S')-6-/‘cr/‘- Buty I- 1 -cy a no-3- [4-(fluoromethyl)- 1 ,3-oxazol-2-yl] -8-oxo- 19-oxa-4,7 - diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-9-yl]-2-hydroxy-3- methylbutanamide (DZA-166). Compound was prepared according to General procedure C from macrocycle 28 (100 mg; 0.16 mmol), TFA (122 pL; 1.59 mmol; 10 equiv.) in DCM (1 mL) in 1 h and 5-HzVA (28 mg; 0.24 mmol; 1.5 equiv.), EDC^XHC1 (61 mg; 0.32 mmol; 2 equiv.), HOBt (64 mg; 0.48 mmol; 3 equiv.), DIPEA (137 pL; 0.79 mmol; 5 equiv.) in DMF (2 mL) in 1 h. Product DZA-166 was purified by reverse phase flash chromatography (10% to 50% MeCN in 0.01% TFA in water) to afford 70 mg (70%) as a white amorphous solid.

’H NMR (400 MHz, MeOD) 5 8.35 (d, J= 7.6 Hz, 1H), 8.24 (d, J= 4.9 Hz, 1H), 7.40 - 7.26 (m, 2H), 6.98 (s, 1H), 5.45 (s, 1H), 5.33 (s, 1H), 4.76 - 4.69 (m, 1H), 4.55 (dd, J= 11.8, 3.8 Hz, 1H), 3.86 (d, J= 3.8 Hz, 1H), 3.28 - 3.06 (m, 3H), 3.02 - 2.82 (m, 3H), 2.09 (septd, J= 6.8, 3.8 Hz, 1H), 1.01 (s, 9H), 1.00 (d, .7= 6.8 Hz, 3H), 0.89 (d, = 6.8 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.8, 174.0,

164.2, 156.1, 149.9, 142.8, 142.5, 140.7 (d, J = 8.2 Hz), 139.2 (d, J = 19.7 Hz), 136.8, 131.8, 129.7,

128.2, 126.6, 119.5, 77.4, 76.6 (d, J= 163.7 Hz), 59.8, 56.3, 47.7, 39.3, 39.2, 34.4, 33.2, 31.8, 26.8, 19.5, 16.3. HRMS (m/z): C30H35N5O5F [M+H]⁺ found: 564.2625. Calculated: 564.2622. [a]_D ²⁰ -155 (c 1.0, MeOH).

Butyl A-[(15',65',95)-6-terCbutyl-l-cyano-3-[4-(difluoromethyl)-l,3-oxazol-2-yl]-8-oxo-19- oxa-4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-9-yl]carbamate (29). To a solution of alcohol 26 (476 mg; 0.85 mmol) in dry DCM (5 mL) DMP (395 mg; 0.93 mmol, 1.1 equiv.) was added in one portion. The resulting suspension was stirred at room temperature for 1 h. Then the suspension was diluted with EtOAc and washed with aqueous IM Na2S2O₃ and aqueous saturated NaHCO₃. Organic layer was washed with brine, dried (Na2SO4), evaporated. The crude aldehyde was used in the next step without additional purification. To a crude aldehyde (474 mg; 0.85 mmol) in dry DCM (2 mL) DAST (491 pL; 3.81 mmol; 4.5 equiv.) was added after the flask was cooled to -78°C in dry ice-acetone bath. The resulting solution was stirred for 30 minutes at the same temprature and then warmed to room temperature and stirred for 2 h. Then the solution was diluted with EtOAc and washed with aqueous saturated NaHCO₃ (^x2). The organic layer was washed with brine, dried (Na2SO4), evaporated. The resulting yellow amorphous material was purified with column chromatography (30% to 50% EtOAc in hexanes) to give difluoride 29 (154 mg; 31%) as a white amorphous material.

’H NMR (400 MHz, MeOD) 5 8.42 (t, J= 2.5 Hz, 1H), 7.38 - 7.25 (m, 2H), 7.02 (s, 1H), 6.89 (s, J = 54 Hz, 1H), 4.79 - 4.71 (m, 1H), 4.14 (dd, J= 11.9, 3.7 Hz, 1H), 3.29 - 3.19 (m, 1H), 3. 19 - 3.03 (m, 2H), 3.01 - 2.80 (m, 3H), 1.44 (s, 9H), 1.03 (s, 9H). ¹³C NMR (101 MHz, MeOD) 5 174.8, 164.4,

157.2, 156.6, 150.2, 142.6, 142.2, 140.5 (t, J= 6.7 Hz), 138.3 (t, J= 27.8 Hz), 137.3, 131.9, 129.7,

127.8, 126.5, 119.5, 111.2 (t, J= 234.1 Hz), 80.6, 59.5, 58.1, 47.7, 39.1, 38.8, 34.3, 31.7, 28.7, 26.8. HRMS (m/z): C₃₀H₃₃N₅O₅F2Na [M+Na]⁺ found: 604.2371. Calculated: 604.2347. [a]_D ²⁰ -127 (c 1.0, MeOH).

(2S)-N- [( LS.6.S.9.S )-6-/‘cr/‘-bu ty I- 1 -cy a no-3- [4-(difluoromethyl)- 1 ,3-oxazol-2-yl] -8-oxo- 19-oxa- 4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-9-yl]-2-hydroxy-3- methylbutanamide (DZA-174). Compound was prepared according to General procedure C from macrocycle 29 (120 mg; 0.21 mmol), TFA (158 pL; 2.06 mmol; 10 equiv.) in DCM (1 mL) in 2 h and 5-HzVA (37 mg; 0.31 mmol; 1.5 equiv.), EDC^XHC1 (119 mg; 0.62 mmol; 3 equiv.), HOBt (84 mg; 0.62 mmol; 3 equiv.), DIPEA (179 pL; 1.03 mmol; 5 equiv.) in DMF (2 mL) in 1 h. Product DZA-174 was purified by reverse phase flash chromatography (10% to 50% MeCN in 0.01% TFA in water) to afford 70 mg (58%) as a white amorphous solid.

^XH NMR (400 MHz, MeOD) 5 8.42 (t, J= 2.5 Hz, 1H), 8.35 (d, J= 7.6 Hz, 1H), 7.38 - 7.30 (m, 2H), 7.00 (s, 1H), 6.90 (t, J = 54.2 Hz, 1H), 4.76 - 4.70 (m, 1H), 4.55 (dd, J= 11.8, 3.8 Hz, 1H), 3.86 (d, J= 3.8 Hz, 1H), 3.29 - 3.07 (m, 3H), 3.01 - 2.86 (m, 3H), 2.09 (septd, J= 6.9, 3.8 Hz, 1H), 1.01 (s, 9H), 1.00 (d, J= 6.8 Hz, 3H), 0.89 (d, J= 6.8 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.8, 174.0,

164.2, 156.6, 150.3, 142.8, 142.4, 140.5 (t, J= 6.7 Hz), 138.3 (t, J= 27.8 Hz), 136.8, 131.9, 129.7,

127.9, 126.6, 119.4, 111.2 (t, J = 234 Hz), 76.83, 59.73, 56.31, 47.73, 39.22, 39.16, 34.36, 33.17, 31.75, 26.75, 19.53, 16.34. HRMS (m/z): C30H34N5O5F2 [M+H]⁺ found: 582.2537. Calculated: 582.2528. [a]_D ²⁰ -163 (c 1.0, MeOH).

Methyl 5-bromo-2-[( 1S)-l-[(2S)-2-{[(tert-butoxy)carbonyl]amino}-3-(3-cyano-2,3-dihydro-lH- inden-5-yl)propanamido]-2,2-dimethylpropyl]-l,3-oxazole-4-carboxylate (31). Compound was prepared according to General procedure A from ester 3 (460 mg; 1.34 mmol), LiOHxH2kO (280 mg; 6.68 mmol; 5 equiv.) in THF/water (1: 1 v/v; 10 mL) and amine 30 (389 mg; 1.34 mmol; 1 equiv.), EDC^XHC1 (384 mg; 2.00 mmol; 1.5 equiv.) in pyridine (4 mL) in 1 h. Product 31 was purified by direct phase flash chromatography (from 20% to 70% EtOAc in hexanes) to afford 476 mg (59%) of a 1 : 1 mixture of diastereomers as a white solid.

‘H NMR (400 MHz, CDCI3) 8 7.24 (s, 0.5H), 7.23 (s, 0.5H), 7.15 - 7.04 (m, 2H), 6.75 (d, J= 8.2 Hz, 0.5H), 6.68 (d, J= 8.2 Hz, 0.5H), 5.06 - 4.97 (m, 2H), 4.33 - 4.24 (m, 1H), 4.10 - 3.98 (m, 1H), 3.93 (s, 1.5H), 3.92 (s, 1.5H), 3.12 - 2.95 (m, 3H), 2.93 - 2.80 (m, 1H), 2.58 - 2.48 (m, 1H), 2.38 - 2.27 (m, 1H), 1.42 (s, 9H), 0.94 (s, 4.5H), 0.93 (s, 4.5H). ¹³C NMR (101 MHz, CDCI3) 6 170.99, 170.97, 164.02, 163.99, 160.9, 155.6, 141.7, 138.2, 136.1, 136.0, 130.49, 130.47, 129.8, 129.6, 128.3, 125.4, 125.2, 125.1, 121.1, 121.0, 80.6, 56.1, 55.8, 55.7, 52.54, 52.51, 37.2, 35.9, 35.8, 34.5, 31.4, 31.17, 31.15, 28.4, 26.3. HRMS (m/z): C28H₃5N₄O₆BrNa [M+Na]⁺ found: 625.1649. Calculated: 625.1638.

Methyl (15',65',95)-9-{[(tert-butoxy)carbonyl]amino}-6-tert-butyl-l-cyano-8-oxo-19-oxa-4,7- diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaene-3-carboxylate (32). Compound was prepared according to General procedure B from amide 31 (476 mg; 0.79 mmol) and K₃PO₄ (837 mg; 3.94 mmol; 5 equiv.) in DMSO (15 mL) in 5 h. Product 32 was purified by reverse phase flash chromatography (from 10% to 70% MeCN in 0,01% TFA in water) to afford 385 mg (93%; dr 99: 1) of a single diastereomer as a white amorphous solid.

‘HNMR (400 MHz, CDCh) 6 7.26 - 7.22 (m, 2H), 7.06 (s, 1H), 5.70 (d, J= 7.3 Hz, 1H), 5.23 (d, J = 9.2 Hz, 1H), 4.67 (d, J = 7.3 Hz, 1H), 4.01 (s, 3H), 3.93 (ddd, J = 12.1, 8.1, 3.3 Hz, 1H), 3.35 - 3.25 (m, 1H), 3.21 (dd, J= 12.1, 12.1 Hz, 1H), 3.18 - 3.10 (m, 1H), 3.01 - 2.92 (m, 1H), 2.87 (dd, J = 12.1, 3.4 Hz, 1H), 2.60 (dt, J = 13.5, 8.2 Hz, 1H), 1.42 (s, 9H), 0.96 (s, 9H). ¹³C NMR (101 MHz, CDCh) 6 171.8, 161.2, 160.7, 155.3, 153.5, 141.5, 140.5, 135.5, 130.8, 130.2, 129.8, 125.4, 118.3, 80.5, 58.3, 58.2, 52.5, 46.7, 39.1, 37.8, 33.6, 31.1, 28.4, 26.6. HRMS (m/z): C28H₃4N₄O₆Na [M+Na]⁺ found: 545.2369. Calculated: 545.2376. [a]_D ²⁰ -116 (c 1.0, MeOH). tert-Butyl -|( 15',65',95)-6-tert-butyl-l-cyano-8-oxo-3-[(l,l,l-trifluoro-3-hydroxypropan-2- yl)carbamoyl]-19-oxa-4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-9- yljcarbamate (33). Methyl ester 32 (385 mg; 0.74 mmol) was dissolved in THF (4 mL). Separately solid LiOHxH2O (93 mg; 0.2.21 mmol; 3 equiv.) was dissolved in water (2 mL) and added to the reaction mixture. Emulsion was stirred at room temperature for 30 min, then aqueous IN HC1 was added. The resulting mixture was extracted with EtOAc (x2), combined organic layers were washed with brine, dried (NfeSCh) and evaporated. The crude carboxylic acid was used in the next step without additional purification. The off-white amorphous solid was dissolved in anhydrous DMF (5 mL) and 2-amino-3,3,3-trifhioropropan-l-ol hydrochloride (183 mg; 1.11 mmol; 1.5 equiv.), EDOEICI (212 mg; 1.11 mmol; 1.5 equiv.) and HOBt (199 mg; 1.47 mmol; 2 equiv.) were added followed by DIPEA (382 pL; 2.21 mmol; 3 equiv.). The solution was stirred at room temperature for

1 h. The solution was diluted with aqueous saturated NEUC1 and EtOAc. The layers were separated and the organic phase was washed with brine, dried (Na2SO4) and evaporated. Product 33 was purified by reverse phase flash chromatography (10% to 70% MeCN in 0.01% TFA in water) to afford 257 mg (56%) as a white amorphous solid.

'H NMR (400 MHz, CDCh) 8 7.54 (d, J= 9.7 Hz, 1H), 7.25 - 7.21 (m, 2H), 7.12 (s, 1H), 5.95 (d, J = 7.7 Hz, 1H), 5.41 (d, J = 9.3 Hz, 1H), 4.96 - 4.77 (m, 1H), 4.66 (d, J= 7.7 Hz, 1H), 4.09 (dd, J= 12.1, 3.9 Hz, 1H), 3.98 - 3.85 (m, 2H), 3.30 - 3.17 (m, 2H), 3.16 - 3.05 (m, 1H), 2.97 - 2.80 (m, 3H), 2.70 (brs, 1H), 1.42 (s, 9H), 0.97 (s, 9H). ¹³C NMR (101 MHz, CDCh) 6 172.2, 160.3, 160.2, 155.4, 151.3, 141.6, 140.5, 135.4, 131.8, 130.3, 129.6, 125.6, 124.7 (q, J = 282.5 Hz), 118.4, 80.6, 59.7, 58.3, 57.8, 51.3 (q, J = 29.7 Hz), 46.5, 38.2, 37.8, 33.5, 31.0, 28.4, 26.5. HRMS (m/z): C₃₀H₃₆N₅O6F₃Na [M+Na]⁺ found: 642.2545. Calculated: 642.2515. tert- Butyl A-[(15',65',95)-6-terCbutyl-l-cyano-8-oxo-3-[4-(trifluoromethyl)-l,3-oxazol-2-yl]-19- oxa-4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-9-yl]carbamate (34). To a stirred solution of amide 33 (240 mg; 0.39 mmol) in DCM (3 mL) was added NaHCO₃ (65 mg; 0.78 mmol; 2 equiv.) followed by DMP (197 mg; 0.47 mmol). The reaction mixture was stirred at room temperature for 1 h before being quenched with aqueous saurated NaHCO₃ (5 mL) and Na2S2O₃ (5 mL). The mixture was stirred vigorously for 1 h before being extracted with DCM (x2). The combined organic extracts were then dried (Na2SO4) and evaporated to afford a crude aldehyde, which was used without further purification. To a stirred solution of the crude aldehyde in DCM (3 mL) at 0 °C was added sequentially DIPEA (268 pL, 1.55 mmol; 4 equiv.), PPh₃ (203 mg, 0.78 mmol;

2 equiv.) and Br2C2C14 (252 mg, 0.78 mmol; 2 equiv.). The reaction mixture was allowed to warm to room temperature and stirred for 1 h before being recooled to 0°C and DBU (231 pL, 1.55 mmol; 4 equiv.) added. The reaction mixture was then stirred for 2 h at room temperature before being quenched with H2O and extracted with DCM (x2). The combined organic extracts were then dried (Na2SO4) and evaporated. The resulting orange oil was purified with direct phase flash chromatography (10% to 50% EtOAc in hexanes) to give bioxazole 34 (25 mg; 11%) as a white amorphous solid.

‘HNMR (400 MHz, CDCh) 6 8.13 (q, J= 1.4 Hz, 1H), 7.26 - 7.22 (m, 2H), 7.10 (s, 1H), 5.79 (d, J = 7.7 Hz, 1H), 5.25 (d, J = 92 Hz, 1H), 4.78 (d, J= 7.7 Hz, 1H), 3.92 (ddd, J = 12.3, 9.1, 3.4 Hz, 1H), 3.34 - 3.12 (m, 3H), 3.02 - 2.91 (m, 1H), 2.90 - 2.78 (m, 2H), 1.43 (s, 9H), 1.00 (s, 9H). HRMS (m/z): C₃oH32N505F₃Na [M+Na]⁺ found: 622.2245. Calculated: 622.2253.

(2S)-N- [( 15',65',95)-6-terCButyl- l-cyano-8-oxo-3- [4-(trifluoromethyl)- 1 ,3-oxazol-2-yl] - 19-oxa- 4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-9-yl]-2-hydroxy-3- methylbutanamide (DZA-175). Compound was prepared according to General procedure C from macrocycle 34 (23 mg; 0.038 mmol), TFA (30 pL; 0.38 mmol; 10 equiv.) in DCM (0.5 mL) in 2 h and 5-HzVA (7 mg; 0.058 mmol; 1.5 equiv.), EDC^XHC1 (22 mg; 0.12 mmol; 3 equiv.), HOBt (16 mg; 0.12 mmol; 3 equiv.), DIPEA (33 pL; 0.19 mmol; 5 equiv.) in DMF (0.5 mL) in 1 h. Product DZA-175 was purified by reverse phase flash chromatography (10% to 50% MeCN in 0.01% TFA in water) to afford 10 mg (44%) as a white amorphous solid.

¹H NMR (400 MHz, MeOD) 5 8.70 (q, J= 1.5 Hz, 1H), 8.36 (d, J= 7.5 Hz, 1H), 8.01 (d, = 8.5 Hz, 1H), 7.39 - 7.31 (m, 2H), 6.99 (s, 1H), 4.73 (d, J= 7.5 Hz, 1H), 4.61 - 4.51 (m, 1H), 3.86 (d, J= 3.8 Hz, 1H), 3.29 - 3.08 (m, 3H), 3.05 - 2.85 (m, 3H), 2.08 (sept d, J= 8.4, 3.8 Hz, 1H), 1.01 (s, 9H), 1.00 (d, .7= 6.8 Hz, 3H), 0.90 (d, = 6.8 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.8, 174.0, 164.3, 157.0, 150.9, 142.9, 142.3, 141.8 (q, J= 4.2 Hz), 136.8, 134.3 (q, J= 40.2 Hz), 131.9, 129.8, 127.6, 126.6, 122.0 (q, J= 266.5 Hz), 119.3, 76.8, 59.7, 56.3, 47.8, 39.3, 39.1, 34.4, 33.2, 31.8, 26.8, 19.5, 16.3. HRMS (m/z): C30H33N5O5F3 [M+H]⁺ found: 600.2433. Calculated: 600.2434. [a]_D ²⁰ -141 (c 1.0, MeOH).

The synthesis of diazonamide A analogs DZA-146, DZA-150, DZA-151, DZA-152 and DZA-153 is shown in Scheme 9.

Amide analogs DZA-151, DZA-146 and DZA-153 and amine analogs DZA-150 and DZA-152 have been prepared as follows. Morpholine moiety containing amide was prepared from macrocycle 6. In the first step methyl ester was hydrolized and then coupled with corresponding morpholine in the presence of EDC hydrochloride, HOBt and DMAP. In the second step the resulting amide 35 was converted into analog DZA-151 in the two-step transformation. Amines DZA-150 and DZA-152 were synthesized from alcohol 26. After alcohol mesylation reaction followed SN2 process in order to substitute mesylate to corresponding amine under basic conditions. Amine 36 was prepared from dimethylamine and amine 37 was prepared from piperidine. Then after A-Boc deprotection and coupling with 5-HzVA 7 analogs DZA-150 and DZA-152 were furnished. Amides DZA-146 and DZA-153 were prepared from analog DZA-129 in one-step amynolysis process with corresponding ammonia or dimethyl amine (Scheme 9).

Scheme 9. Synthesis of diazonamide A analogs DZA-146, DZA-150, DZA-151, DZA-152 and

DZA-153, tert- Butyl V-[( LS.6.S.9. )-6-/‘ r/‘-butyl-l-cy:ino-3-|4-(inorpholine-4-carbonyl)-l ,3-oxazol-2-yl]-8- oxo-19-oxa-4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-9-yl]carbamate

(35). Methyl ester 6 (120 mg; 0.20 mmol) was dissolved in THF (1 mL). Separately solid LiOHxH^O (26 mg; 0.61 mmol; 3 equiv.) was dissolved in water (0.5 mL) and added to the reaction mixture. Emulsion was stirred at room temperature for 30 min, then aqueous IN HC1 was added. The resulting mixture was extracted with EtOAc (x2), combined organic layers were washed with brine, dried (Na2SO4) and evaporated. The crude carboxilyc acid was used in the next step without additional purification. To crude carboxylic acid (117 mg; 0.20 mmol) in DMF (1 mL) EDOHCl (78 mg; 0.41 mmol; 2 equiv.), HOBt (82 mg; 0.61 mmol; 3 equiv.) and morpholine (88 pL; 1.02 mmol; 5 equiv.) were added at room temperature followed by DIPEA (352 pL; 2.03 mmol; 10 equiv.). The resulting yellow solution was stirred for 20 h at room temperature and then diluted with EtOAc and washed with aqueous saturated NH4CI, water and brine. Organic layer were dried (Na2SO4) and evaporated. The resulting orange oil was purified with reverse phase flash chromatography (10% to 60% MeCN in 0.01% TFA in water) to give amide 35 (70 mg; 63%) as a white amorphous solid. ’H NMR (400 MHz, CDCh) 6 8.33 (s, 1H), 7.26 (s, 2H, overlapping with CDCh), 7.11 (s, 1H), 5.81 (d, J= 7.5 Hz, 1H), 5.23 (d, J= 9.3 Hz, 1H), 4.73 (d, J= 7.4 Hz, 1H), 4.39 - 4.22 (m, 2H), 3.95 (ddd, J= 12.4, 9.3, 3.4 Hz, 1H), 3.85 - 3.71 (m, 6H), 3.36 - 3.19 (m, 2H), 3.09 (ddd, = 13.1, 8.3, 4.6 Hz, 1H), 2.98 (ddd, J= 15.8, 8.3, 4.6 Hz, 1H), 2.89 (dd, J= 12.4, 3.4 Hz, 1H), 2.80 - 2.71 (m, 1H), 1.43 (s, 9H), 0.99 (s, 9H). ¹³C NMR (101 MHz, CDCh) 8 171.9, 161.6, 160.4, 155.4, 153.3, 148.2, 144.4,

141.4, 140.5, 138.3, 135.7, 130.4, 129.8, 127.6, 125.5, 118.5, 80.6, 67.5, 67.0, 58.3, 47.4, 46.4, 43.2, 38.8, 37.9, 33.6, 31.1, 28.4, 26.6. HRMS (m/z): C34H41N6O7 [M+H]⁺ found: 645.3036. Calculated:

645.3037. [a]_D ²⁰ -119 (c 1.0, CHCh).

(25)-N-[(15',65',95)-6-terCbutyl-l-cyano-3-[4-(morpholine-4-carbonyl)-l,3-oxazol-2-yl]-8-oxo- 19-oxa-4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-9-yl]-2-hydroxy-3- methylbutanamide (DZA-151). Compound was prepared according to General procedure C from macrocycle 35 (60 mg; 0.094 mmol), TFA (72 pL; 0.93 mmol; 10 equiv.) in DCM (0.5 mL) in 1 h and 5-HzVA (17 mg; 0.14 mmol; 1.5 equiv.), EDC^XHC1 (36 mg; 0.19 mmol; 2 equiv.), HOBt (38 mg; 0.28 mmol; 3 equiv.), DIPEA (81 pL; 0.47 mmol; 5 equiv.) in DMF (1 mL) in 1 h. Product DZA- 151 was purified by reverse phase flash chromatography (10% to 50% MeCN in 0.01% TFA in water) to afford 50 mg (83%) as a white amorphous solid.

^XH NMR (400 MHz, MeOD) 6 8.55 (s, 1H), 7.40 - 7.33 (m, 2H), 6.96 (s, 1H), 4.66 (s, 1H), 4.59 (dd, J= 11.8, 3.9 Hz, 1H), 4.26 (s, 2H), 3.83 - 3.72 (m, 6H), 3.36 - 3.26 (m, 1H), 3.18 (dd, J= 12.1, 12.1 Hz, 1H), 3.14 - 3.07 (m, 1H), 3.06 - 2.96 (m, 1H), 2.91 (dd, J = 12.4, 3.9 Hz, 1H), 2.88 - 2.79 (m, 1H), 2.09 (sept d, J = 6.9, 3.9 Hz, 1H), 1.01 (d, J = 6.8 Hz, 3H), 1.00 (s, 9H), 0.90 (d, J= 6.8 Hz, 3H). ¹³C NMR (101 MHZ, MeOD) 6 175.8, 173.9, 164.0, 162.2, 154.9, 150.2, 145.6, 143.0, 142.1, 138.7, 136.8, 131.9, 130.0, 128.1, 126.6, 119.7, 76.8, 68.3, 67.8, 60.0, 56.3, 47.8, 44.3, 39.7, 39.2,

34.4, 33.2, 31.8, 26.8, 19.5, 16.3. HRMS (m/z): C34H41N6O7 [M+H]⁺ found: 645.3039. Calculated:

645.3037. [a]_D ²⁰ -139 (c 1.0, MeOH). terf-Butyl N-[(15',65',95)-6-terCbutyl-l-cyano-3-{4-[(dimethylamino)methyl]-l,3-oxazol-2-yl}-8- oxo-19-oxa-4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-9-yl]carbamate

(36). To alcohol 26 (265 mg; 0.47 mmol) in dry THF (4 mL) TEA (132 pL; 0.94 mmol; 2 equiv.) was added dropwise followed by DMAP (1 mg; 0.009 mmol; 0.02 equiv.) and the resulting solution was cooled to 0°C in ice bath. Then MsCl (55 pL; 0.71 mmol; 1.5 equiv.) was added dropwise to the solution and the resulting orange solution was stirred at the same temperature for 30 minutes. Then the solution was diluted with EtOAc and washed with IN HC1, water and brine. Organic layer was dried (Na2SO4) and evaporated. The crude mesylate was used in the next step without additional purification. To crude meslate (145 mg; 0.23 mmol) in dry THF (1 mL) TEA (158 pL; 1.13 mmol; 5 equiv.) was added dropwise at room temperature followed by Me2NH><HCl (92 mg; 1.13 mmol; 5 equiv.). The resulting yellow solution was stirred for 20 h, white precipitate was formed. The suspension was diluted with EtOAc and washed with aqueous saturated NH4CI, water and brine. Organic layer was dried (Na2SO4) and evaporated. The resulting yellow oil was purified with reverse phase flash chromatography (10% to 70% MeCN in 0.01% TFA in water) to give amine 36 (63 mg; 47%) as a white amorphous solid.

’H NMR (400 MHz, MeOD) 5 8.34 (s, 1H), 8.20 (d, J = 7.2 Hz, 1H), 7.38 - 7.31 (m, 2H), 6.88 (s, 1H), 4.71 - 4.65 (m, 1H), 4.45 (d, J= 19.7 Hz, 1H), 4.41 (d, J= 19.7 Hz, 1H), 4.17 (dd, J= 11.8, 3.7 Hz, 1H), 3.34 - 3.26 (m, 1H), 3.18 - 3.03 (m, 3H), 3.01 (s, 6H), 2.90 - 2.76 (m, 2H), 1.44 (s, 9H), 1.01 (s, 9H). ¹³C NMR (101 MHZ, MeOD) 5 174.8, 164.5, 157.2, 156.7, 150.5, 142.8, 142.2, 137.3, 133.4, 131.9, 129.7, 128.2, 126.5, 119.7, 80.7, 60.2, 60.1, 58.1, 52.8, 47.8, 43.4, 39.9, 38.7, 34.4, 31.8, 28.7, 26.8. HRMS (m/z): C32H41N6O5 [M+H]⁺ found: 589.3149. Calculated: 589.3138. [a]_D ²⁰ - 95 (c 1.0, MeOH).

(2S)-N- [( LS.6.S.9.S)-6-/‘c/7‘- Bu ty I- l-cyano-3- {4- [(dimethylamino)methyl] - 1 ,3-oxazol-2-yl}-8-oxo- 19-oxa-4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-9-yl]-2-hydroxy-3- methylbutanamide (DZA-150). Compound was prepared according to General procedure C from macrocycle 36 (40 mg; 0.068 mmol), TFA (52 pL; 0.68 mmol; 10 equiv.) in DCM (0.5 mL) in 1 h and 5-HzVA (12 mg; 0.10 mmol; 1.5 equiv.), EDC^XHC1 (26 mg; 0.14 mmol; 2 equiv.), HOBt (27 mg; 0.20 mmol; 3 equiv.), DIPEA (58 pL; 0.34 mmol; 5 equiv.) in DMF (1 mL) in 1 h. Product DZA- 150 was purified by reverse phase flash chromatography (10% to 50% MeCN in 0.01% TFA in water) to afford 23 mg (58%) as a white amorphous solid.

’H NMR (400 MHz, MeOD) 5 8.37 (d, J= 7.2 Hz, 1H), 8.34 (s, 1H), 7.40 - 7.34 (m, 2H), 6.86 (m, 1H), 4.68 - 4.64 (m, 1H), 4.58 (dd, J= 11.7, 3.9 Hz, 1H), 4.44 (d, J= 19.9 Hz, 1H), 4.41 (d, J= 19.9 Hz, 1H), 3.87 (d, J= 3.8 Hz, 1H), 3.36 - 3.27 (m, 1H, overlapping with MeOD), 3.20 - 3.11 (m, 2H), 3.09 - 3.03 (m, 1H), 3.01 (s, 6H), 2.90 (dd, J= 12.4, 3.8 Hz, 1H), 2.84 - 2.75 (m, 1H), 2.09 (sept d, J = 6.9, 3.8 Hz, 1H), 1.01 (d, J = 6.8 Hz, 3H) 1.00 (s, 9H), 0.89 (d, J = 6.8 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.8, 174.0, 164.4, 156.6, 150.6, 143.0, 142.8, 142.3, 136.9, 133.4, 131.9, 129.7, 128.3, 126.6, 119.7, 76.8, 60.2, 56.2, 52.8, 47.8, 43.4, 40.0, 39.1, 34.4, 33.2, 31.9, 26.8, 19.5, 16.3. HRMS (m/z): C32H41N6O5 [M+H]⁺ found: 589.3143. Calculated: 589.3138. [a]_D ²⁰ -134 (c 1.0, MeOH). terf-Butyl /V-[(LS,6»S,9»S)-6-terCbutyl-l-cyano-8-oxo-3-{4-[(piperidin-l-yl)methyl]-l,3-oxazol-2- yl}-19-oxa-4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-9-yl]carbamate

(37). To alcohol 26 (70 mg; 0.13 mmol) in dry THF (1 mL) TEA (35 pL; 0.25 mmol; 2 equiv.) was added dropwise followed by DMAP (3 mg; 0.03 mmol; 0.2 equiv.) and the resulting solution was cooled to 0°C in ice bath. Then MsCl (15 pL; 0.19 mmol; 1.5 equiv.) was added dropwise to the solution and the resulting orange solution was stirred at the same temperature for 30 minutes. Then the solution was diluted with EtOAc and washed with IN HC1, water and brine. Organic layer was dried (Na2SO4) and evaporated. The crude mesylate was used in the next step without additional purification. To crude meslate (80 mg; 0.13 mmol) in dry DMF (1 mL) K2CO3 (86 mg; 0.63 mmol; 5 equiv.) was added in one portion at room temperature followed by piperidine (124 pL; 1.25 mmol; 10 equiv.). The resulting yellow suspesnion was stirred for 1 h. The suspension was diluted with EtOAc and washed with aqueous IN HC1 (x2) and brine. Organic layer was dried (Na2SO4) and evaporated. The resulting yellow oil was purified with reverse phase flash chromatography (10% to 70% MeCN in 0.01% TFA in water) to give amine 37 (40 mg; 51%) as a white amorphous solid.

'H NMR (400 MHz, MeOD) 5 8.33 (s, 1H), 8.20 (d, J= 7.1 Hz, 1H), 7.42 - 7.30 (m, 2H), 6.86 (d, J = 1.2 Hz, 1H), 4.70 - 4.64 (m, 1H), 4.45 - 4.34 (m, 2H), 4.17 (dd, J= 11.9, 3.7 Hz, 1H), 3.76 - 3.69 (m, 1H), 3.66 - 3.60 (m, 1H), 3.33 - 3.26 (m, 1H, overlapping with MeOD), 3.21 - 3.10 (m, 3H), 3.08 - 2.99 (m, 2H), 2.87 (dd, J= 12.3, 3.6 Hz, 1H), 2.82 - 2.74 (m, 1H), 2.01 - 1.94 (m, 2H), 1.86 - 1.75 (m, 3H), 1.55 - 1.50 (m, 1H), 1.44 (s, 9H), 1.01 (s, 9H). ¹³C NMR (101 MHz, MeOD) 5 174.8, 164.5, 157.2, 156.6, 150.4, 142.8, 142.2, 137.3, 133.1, 131.9, 129.8, 128.2, 126.6, 119.8, 80.7, 60.1, 58.1, 54.4, 52.1, 47.8, 40.0, 38.7, 34.4, 31.8, 28.7, 26.8, 24.4, 22.4. HRMS (m/z): C35H45N6O5 [M+H]⁺ found: 629.3452. Calculated: 629.3451. [a]_D ²⁰ -97 (c 1.0, MeOH).

(2S)-N-[( l.S.6.S.9.S)-6-/‘cr/‘-butyl-l-cyano-8-oxo-3-14-|(piperidin-l-yl)niethyl|-l .3-oxazol-2-ylJ- 19-oxa-4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-9-yl]-2-hydroxy-3- methylbutanamide (DZA-152). Compound was prepared according to General procedure C from macrocycle 37 (38 mg; 0.06 mmol), TFA (46 pL; 0.60 mmol; 10 equiv.) in DCM (0.5 mL) in 1 h and 5-HzVA (11 mg; 0.091 mmol; 1.5 equiv.), EDC^XHC1 (23 mg; 0.12 mmol; 2 equiv.), HOBt (25 mg; 0.18 mmol; 3 equiv.), DIPEA (52 pL; 0.30 mmol; 5 equiv.) in DMF (0.5 mL) in 1 h. Product DZA- 152 was purified by reverse phase flash chromatography (10% to 50% MeCN in 0.01% TFA in water) to afford 20 mg (53%) as a white amorphous solid.

’H NMR (400 MHz, MeOD) 5 8.33 (s, 1H), 7.40 - 7.33 (m, 2H), 6.85 (s, 1H), 4.66 - 4.63 (m, 1H), 4.59 (dd, J= 11.8, 3.9 Hz, 1H), 4.41 (d, J= 23.3 Hz, 1H), 4.37 (d, J= 23.3 Hz, 1H), 3.87 (d, J= 3.9 Hz, 1H), 3.73 (d, J= 12.3 Hz, 1H), 3.63 (d, J= 12.0 Hz, 1H), 3.36 - 3.28 (m, 1H, overlapping with MeOD), 3.22 - 3.11 (m, 3H), 3.10 - 3.00 (m, 2H), 2.90 (dd, J= 12.5, 3.9 Hz, 1H), 2.83 - 2.74 (m, 1H), 2.09 (sept d, J= 6.9, 3.9 Hz, 1H), 2.01 - 1.93 (m, 2H), 1.86 - 1.72 (m, 3H), 1.59 - 1.47 (m, 1H), 1.01 (d, .7= 6.8 Hz, 3H), 1.00 (s, 9H), 0.89 (d, = 6.8 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.8, 174.0, 164.3, 156.5, 150.5, 143.0, 142.8, 142.4, 136.9, 133.2, 131.9, 129.8, 128.3, 126.6, 119.7, 76.8, 60.2, 56.2, 54.4, 52.1, 47.8, 40.1, 39.1, 34.4, 33.2, 31.9, 26.8, 24.2, 22.4, 19.5, 16.3. HRMS (m/z): C35H45N6O5 [M+H]⁺ found: 629.3464. Calculated: 629.3451. [a]_D ²⁰ -115 (c 1.0, MeOH).

2- [( 15,6$,95)-6-terCButyl- l-cyano-9- [(25)-2-hydroxy-3-methylbutanamido] -8-oxo- 19-oxa-4,7 - diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-3-yl]-l,3-oxazole-4- carboxamide (153). To a solution of ester DZA-129 (56 mg; 0.096 mmol) in dry MeOH (0.5 mL) NH3 (7M in MeOH; 543 pL; 3.80 mmol, 40 equiv.) was added dropwise at room temperature. The yellowish solution was stirred for 20 h, then evaporated. The resulting yellow amorphous material was purified with reverse phase flash chromatography (10% to 40% MeCN in water) to give amide DZA-153 (39 mg; 72%) as a white amorphous material.

’H NMR (400 MHz, MeOD) 5 8.58 (s, 1H), 7.40 - 7.30 (m, 2H), 6.96 (d, J= 1.4 Hz, 1H), 4.71 (s, 1H), 4.56 (dd, J= 11.8, 3.8 Hz, 1H), 3.86 (d, J= 3.8 Hz, 1H), 3.35 - 3.25 (m, 1H, overlapping with MeOD), 3.23 - 3.13 (m, 2H), 3.03 - 2.94 (m, 1H), 2.93 - 2.84 (m, 2H), 2.09 (sept d, J= 6.8, 3.8 Hz, 1H), 1.01 (s, 9H), 1.00 (d, .7= 6.8 Hz, 3H), 0.90 (d, J= 6.8 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.8, 174.0, 164.8, 164.3, 155.5, 150.5, 144.0, 142.9, 142.5, 138.5, 136.8, 131.9, 129.7, 127.9, 126.6, 119.5, 76.8, 59.8, 56.3, 47.8, 39.6, 39.1, 34.4, 33.2, 31.8, 26.8, 19.5, 16.3. HRMS (m/z): C30H35N6O6 [M+H]⁺ found: 575.2632. Calculated: 575.2618. [a]_D ²⁰ -171 (c 1.0, MeOH).

2- [( 15',65',95)-6-tert-Butyl- l-cyano-9- [(25)-2-hydroxy-3-methylbutanamido] -8-oxo- 19-oxa-4,7 - diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-3-yl]-7V-methyl-l,3-oxazole-4- carboxamide (DZA-146). To a solution of ester DZA-129 (36 mg; 0.061 mmol) MeNH2 (33% in EtOH; 320 pL; 3.05 mmol, 50 equiv.) was added dropwise at room temperature. The yellowish solution was stirred for 1 h, then evaporated. The resulting yellow amorphous material was purified with reverse phase flash chromatography (10% to 50% MeCN in water) to give amide DZA-146 (26 mg; 72%) as a white amorphous material.

^XH NMR (400 MHz, MeOD) 5 8.55 (s, 1H), 7.38 - 7.30 (m, 2H), 6.97 (s, 1H), 4.72 (s, 1H), 4.55 (dd, J= 11.8, 3.8 Hz, 1H), 3.86 (d, J = 3.8 Hz, 1H), 3.34 - 3.24 (m, 1H, overlapping with MeOD), 3.23 - 3.14 (m, 2H), 3.01 - 2.92 (m, 1H), 2.96 (s, 3H), 2.92 - 2.85 (m, 2H), 2.09 (sept d, J= 6.9, 3.8 Hz, 1H), 1.01 (s, 9H), 1.00 (d, .7= 6.9 Hz, 3H), 0.89 (d, J= 6.9 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.8, 174.0, 164.3, 163.1, 155.5, 150.4, 143.3, 142.9, 142.5, 138.6, 136.8, 131.9, 129.7, 127.9, 126.6, 119.5, 76.8, 59.7, 56.3, 47.8, 39.5, 39.1, 34.4, 33.2, 31.8, 26.8, 26.2, 19.5, 16.3. HRMS (m/z): C31H37N6O6 [M+H]⁺ found: 589.2775. Calculated: 589.2775. [a]_D ²⁰ -164 (c 1.0, MeOH).

The synthesis of diazonamide A analogs DZA-160 and DZA-162 is shown in Scheme 10. Accordingly, oxime DZA-160 and acetamide DZA-162 were prepared starting from alcohol 26. To synthesize oxime 38 alcohol was oxidized to aldehyde and then in the presence of hydroxyamine was converted into oxime 38. Then, after A-Boc deprotection and coupling with A'-H/VA 7 analog DZA- 160 was furnished. Acetamide DZA-162 was prepared from alcohol 26 in mesylation and futher SN2 reaction with sodium azide to give 39. Azide was then reduced to primary amine. Acylation of amine gave acetamide 40, which was converted into analog DZA-162 in deprotection-amidation reaction sequence (Scheme 10).

Scheme 10. Synthesis of diazonamide A analogs DZA-160 and DZA-162. tert- Butyl V-[( 15,65, 95)-6-terCbutyl-l-cyano-3-{4-[(£)-(hydroxyimino)methyl]-l, 3-oxazol-2- yl}-8-oxo-19-oxa-4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-9- yljcarbamate (38). To a solution of alcohol 26 (225 mg; 0.40 mmol) in dry DCM (2 mL) DMP (187 mg; 0.44 mmol, 1.1 equiv.) was added in one portion. The resulting suspension was stirred at room temperature for 1 h. Then the suspension was diluted with EtOAc and washed with aqueous IM Na2S2C>3 and aqueous saturated NaHCCh. Organic layer was washed with brine, dried (Na2SO4), evaporated. The crude aldehyde was used in the next step without additional purification. To a crude aldehyde (224 mg; 0.40 mmol) in MeOH (2 mL) hydroxylamine HC1 (84 mg; 1.20 mmol, 3 equiv.) was added in one portion followed by K2CO3 (166 mg; 1.20 mmol; 3 equiv.). The resulting suspension was stirred for 2 h at room temperature. The reaction mixture was quenched by the addition of aqueous saturated NH4CI and extracted with EtOAc (x2). Organic layers were combined, washed with brine, dried (Na2SO4) and evaporated. The off-white amorphous solid was purified with reverse phase flash chromatograhy (10% to 50% MeCN in 0.01% TFA in water) to give oxime 38 (85 mg; 37%) as a mixture of E and Z isomers as a white amorphous solid.

’H NMR (400 MHz, MeOD) 5 8.76 (s, 1H), 8.18 (d, J = 7.6 Hz, 1H), 7.54 (s, 1H), 7.36 - 7.27 (m, 2H), 7.02 (s, 1H), 4.79 - 4.74 (m, 1H), 4.20 - 4.09 (m, 1H), 3.29 - 3.20 (m, 1H), 3.20 - 3.02 (m, 2H), 3.02 - 2.80 (m, 3H), 1.44 (s, 9H), 1.03 (s, 9H). ¹³C NMR (101 MHz, MeOD) 5 174.8, 164.3, 157.2,

154.8, 149.8, 144.9, 142.6, 142.3, 139.5, 137.3, 133.6, 131.8, 129.7, 128.0, 126.5, 119.6, 80.6, 59.6, 58.1, 47.7, 39.2, 38.8, 34.3, 31.7, 28.7, 26.8. HRMS (m/z): C30H35N6O6 [M+H]⁺ found: 575.2632. Calculated: 575.2618.

(2.S')-\-|( 15,6»S,9»S)-6-terCButyl-l-cyano-3-{4-[(£)-(hydroxyimino)methyl]-l,3-oxazol-2-yl}-8- oxo-19-oxa-4,7-diazatetracyclo[9.5.2.1², .O¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-9-yl]-2- hydroxy-3-methylbutanamide (DZA-160). Compound was prepared according to General procedure C from macrocycle 38 (62 mg; 0.11 mmol), TFA (166 pL; 2.16 mmol; 20 equiv.) in DCM (1 mL) in 1 h and 5-HzVA (19 mg; 0.16 mmol; 1.5 equiv.), EDC^XHC1 (41 mg; 0.22 mmol; 2 equiv.), HOBt (44 mg; 0.32 mmol; 3 equiv.), DIPEA (93 pL; 0.54 mmol; 5 equiv.) in DMF (2 mL) in 1 h. Product DZA-160 was purified by reverse phase flash chromatography (10% to 50% MeCN in 0.01% TFA in water) to afford 17 mg (27%) as a white amorphous solid.

’H NMR (400 MHz, MeOD) 5 8.77 (s, 1H), 7.55 (s, 1H), 7.43 - 7.26 (m, 2H), 7.00 (s, 1H), 4.74 (s, 1H), 4.55 (dd, J = 11.8, 3.8 Hz, 1H), 3.87 (d, J = 3.8 Hz, 1H), 3.30 - 3.08 (m, 3H), 3.02 - 2.84 (m, 3H), 2.09 (sept d, J = 6.9, 3.8 Hz, 1H), 1.01 (s, 9H), 1.00 (d, J= 6.8 Hz, 3H), 0.90 (d, J = 6.8 Hz, 3H). ¹³C NMR (101 MHZ, MeOD) 5 175.8, 173.9, 164.2, 154.8, 149.9, 144.9, 142.8, 142.5, 139.5,

136.8, 133.6, 131.9, 129.7, 128.1, 126.6, 119.5, 76.8, 59.7, 56.3, 47.7, 39.3, 39.2, 34.4, 33.2, 31.8,

26.8, 19.5, 16.3. HRMS (m/z): C30H35N6O6 [M+H]⁺ found: 575.2621. Calculated: 575.2618. [a]_D ²⁰ - 149 (c 1.0, MeOH). terf-Butyl /V-[(l»S,6»S,9»S)-3-[4-(azidomethyl)-l,3-oxazol-2-yl]-6-terCbutyl-l-cyano-8-oxo-19-oxa- 4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-9-yl]carbamate (39). To alcohol 26 (400 mg; 0.71 mmol) in dry THF (5 mL) TEA (199 pL; 0.142 mmol; 2 equiv.) was added dropwise followed by DMAP (2 mg; 0.014 mmol; 0.02 equiv.) and the resulting solution was cooled to 0°C in ice bath. Then MsCl (83 pL; 1.07 mmol; 1.5 equiv.) was added dropwise to the solution and the resulting orange solution was stirred at the same temperature for 30 minutes. Then the solution was diluted with EtOAc and washed with aqueous IN HC1, water and brine. Organic layer was dried (Na2SO4) and evaporated. The crude mesylate was used in the next step without additional purification. To crude meslate (465 mg; 0.71 mmol) in dry DMF (5 mL) K2CO3 (493 mg; 3.56 mmol; 5 equiv.) was added in one portion at room temperature followed by Nal (11 mg; 0.071 mmol; 0.1 equiv.) and NaNx (139 mg; 2.14 mmol; 3 equiv.). The resulting yellow suspesnion was stirred for 1 h. The suspension was diluted with EtOAc and washed with water and brine. Organic layer was dried (Na2SO4) and evaporated. The resulting yellow oil was purified with direct phase flash chromatography (10% to 80% EtOAc in hexanes) to give azide 39 (180 mg; 50%) as a white amorphous solid.

’H NMR (400 MHz, CDCh) 8 7.79 (s, 1H), 7.25 - 7.21 (m, 2H), 7.12 (s, 1H), 5.78 (d, J= 7.7 Hz, 1H), 5.27 (d, J = 9.3 Hz, 1H), 4.78 (d, J= 7.7 Hz, 1H), 4.41 (d, J = 28.9 Hz, 1H), 4.37 (d, J= 28.9 Hz, 1H), 3.93 (ddd, J= 12.4, 9.3, 3.4 Hz, 1H), 3.33 - 3.20 (m, 2H), 3.20 - 3.11 (m, 1H), 2.99 - 2.93 (m, 1H), 2.93 - 2.82 (m, 2H), 1.43 (s, 9H), 1.00 (s, 9H). ¹³C NMR (101 MHz, CDCh) 6 171.9, 161.7,

155.4, 154.9, 148.1, 141.4, 140.9, 137.7, 136.8, 135.5, 130.2, 129.5, 127.7, 125.5, 118.3, 80.6, 58.3, 58.0, 46.4, 46.3, 38.6, 37.9, 33.5, 31.1, 28.4, 26.6. HRMS (m/z): GoH NxO Na [M+Naf found: 609.2571. Calculated: 609.2550. [a]_D ²⁰ -146 (c 1.0, CHCh). terf-Butyl \-|( LS.6.S.9.S)-6-/‘cr/‘-biityl-l-cyano-3-|4-(acet amidomet hyl)-l .3-oxazol-2-yl|-8-oxo- 19-oxa-4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-9-yl]carbamate (40). To azide 39 (471 mg; 0.80 mmol) in dry MeOH (12 mL) Pd/C (10%; 43 mg; 0.040 mmol; 0.05 equiv.) was added in one portion and H2 was barbotated through the suspension for 1 h at room temperature. Then the suspension was filtered through celite, evaporated. The crude amine was used in the next step without additional purification. To the crude amine (450 mg; 0.80) in DCM (10 mL) TEA (224 pL; 1.61 mmol; 2 equiv.) was added dropwise followed by DMAP (10 mg; 0.08 mmol; 0.1 equiv.) and the flask was cooled to 0°C in ice bath. Then acetic anhydride (76 pL; 0.80 mmol; 1 equiv.) was added dropwise to the solution. The yellow solution was stirred for 10 minutes at the same temperature and 30 minutes at room temperature. The reaction was diluted with EtOAc and washed with saturated aqueous NaHCOa, 1 N HC1, water, and brine. Organic layer was dried (Na2SO4) and evaporated. The resulting yellow oil was purified with direct phase flash chromatography (10% to 50% EtOAc in hexanes) to afford amide 40 (171 mg; 35%) as a white amorphous solid.

’H NMR (400 MHz, CDCh) 6 7.74 (s, 1H), 7.26 - 7.22 (m, 2H), 6.96 (s, 1H), 6.34 (s, 1H), 5.86 (d, J = 6.9 Hz, 1H), 5.23 (d, J = 9.3 Hz, 1H), 4.72 (d, J= 7.3 Hz, 1H), 4.51 (dd, J = 15.1, 5.8 Hz, 1H), 4.35 (dd, J= 15.1, 4.6 Hz, 1H), 3.94 (ddd, = 12.1, 9.3, 3.4 Hz, 1H), 3.38 - 3.28 (m, 1H), 3.24 - 3.14 (m, 2H), 3.01 (ddd, J = 15.6, 8.4, 3.4 Hz, 1H), 2.85 (dd, J = 12.3, 3.4 Hz, 1H), 2.65 (ddd, J = 13.4,

8.4, 8.4 Hz, 1H), 2.00 (s, 3H), 1.43 (s, 9H), 0.98 (s, 9H). ¹³C NMR (101 MHz, CDCh) 5 171.9, 170.6, 162.0, 155.4, 154.5, 148.2, 141.5, 141.1, 138.6, 136.2, 135.6, 130.2, 129.4, 128.1, 125.5, 118.5, 80.6, 58.4, 58.1, 46.6, 39.7, 37.9, 35.6, 33.5, 31.2, 28.4, 26.6, 23.1. HRMS (m/z): C32H39N6O6 [M+H]⁺ found: 603.2939. Calculated: 603.2931. [a]_D ²⁰ -101 (c 1.0, CH2CI2).

(2S)-N- [( l$,6$,9 )-6-terCButyl- l-cyano-3- [4-(acetamidomethyl)- 1 ,3-oxazol-2-yl] -8-oxo- 19-oxa- 4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-9-yl]-2-hydroxy-3- methylbutanamide (DZA-162). Compound was prepared according to General procedure C from macrocycle 40 (171 mg; 0.28 mmol), TFA (436 pL; 5.67 mmol; 20 equiv.) in DCM (2 mL) in 2 h and 5-HzVA (50 mg; 0.43 mmol; 1.5 equiv.), EDC^XHC1 (109 mg; 0.57 mmol; 2 equiv.), HOBt (115 mg; 0.85 mmol; 3 equiv.), DIPEA (246 pL; 1.42 mmol; 5 equiv.) in DMF (3 mL) in 1 h. Product DZA-162 was purified by reverse phase flash chromatography (10% to 50% MeCN in 0.01% TFA in water) to afford 120 mg (70%) as a white amorphous solid.

‘H NMR (400 MHz, MeOD) 5 7.97 (s, 1H), 7.39 - 7.25 (m, 2H), 6.97 (s, 1H), 4.72 (s, 1H), 4.55 (dd, J = 11.8, 3.8 Hz, 1H), 4.38 (s, 2H), 3.86 (d, J= 3.8 Hz, 1H), 3.28 - 3.05 (m, 3H), 3.01 - 2.84 (m, 3H), 2.09 (sept d, J= 6.8, 3.8 Hz, 1H), 2.00 (s, 3H), 1.00 (s, 9H), 1.00 (d, J= 6.8 Hz, 3H), 0.89 (d, J = 6.8 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.8, 173.9, 173.4, 164.1, 155.6, 149.5, 142.8, 142.6, 140.8, 138.3, 136.8, 131.8, 129.7, 128.4, 126.6, 119.6, 76.8, 59.7, 56.3, 47.8, 39.4, 39.2, 36.3, 34.3, 33.2, 31.8, 26.8, 22.5, 19.5, 16.3. HRMS (m/z): C32H39N6O6 [M+H]⁺ found: 603.2955. Calculated: 603.2931. [a]_D ²⁰ -133 (c 1.0, MeOH).

The synthesis of diazonamide A analogs DZA-149 and DZA-161 is shown in Scheme 11.

Accordingly, carbonate 41 was prepared from alcohol 26 after reaction with methylchloroformate under basic conditions. Analog DZA-161 was obtained after 7V-Boc deprotection and amidation reaction sequence. Accordingly, analog DZA-148 was prepared from alcohol 26. After alcohol oxidation to aldehyde it was subjected into Horner-Wadsworth-Emmons reaction to give //-alkene 42. The resulting alkene was converted into analog DZA-148 in two steps. Analog DZA-149 was obtained from analog DZA-148 after alkene Pd-catalyzed hydrogenation (Scheme 11).

Scheme 11. Synthesis of diazonamide A analogs DZA-148-149 and DZA-161,

{2-[( LS,6»S,9»S)-9-{[(tert-Butoxy)carbonyl]amino}-6-tert-butyl-l-cyano-8-oxo-l 9-oxa-4, 7- diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-3-yl]-l,3-oxazol-4-yl}methyl methyl carbonate (41). To alcohol 26 (200 mg; 0.36 mmol) in dry DCM (2 mL) pyridine (32 pL;

0.39 mmol; 1.1 equiv.) was added dropwise at room temperature. Then the flask was cooled to 0°C and methyl chloroformate (50 pL; 0.64 mmol; 1.8 equiv.) was added in one portion. The yellow solution was stirred for 2 h while gradually warming to room temperature. The solution was quenched by the addition of aqueous saturated NH4CI and extracted with EtOAc (x2). Organic layers were combined, washed with brine, dried (Na2SO4) and evaporated. The yellow oil was purified with reverse phase flash chromatograhy (10% to 70% MeCN in 0.01% TFA in water) to give carbonate 41 (145 mg; 66%) as a white amorphous solid.

’H NMR (400 MHz, CDCh) 8 7.86 (s, 1H), 7.25 - 7.21 (m, 2H), 7.11 (s, 1H), 5.77 (d, J = 7.7 Hz, 1H), 5.24 (dd, J= 9.4 Hz, 1H), 5.20 (dd, J = 3.8, 0.9 Hz, 2H), 4.78 (d, J = 7.7 Hz, 1H), 3.95 - 3.87 (m, 1H), 3.81 (s, 3H), 3.33 - 3.19 (m, 2H), 3.19 - 3.11 (m, 1H), 3.00 - 2.90 (m, 1H), 2.89 - 2.78 (m, 2H), 1.43 (s, 9H), 1.00 (s, 9H). ¹³C NMR (101 MHz, CDCh) 6 171.9, 161.7, 155.7, 155.4, 154.6, 148.0, 141.4, 141.0, 138.2, 137.2, 135.5, 130.2, 129.5, 127.7, 125.5, 118.3, 80.6, 61.5, 58.2, 58.0, 55.2, 46.4, 38.7, 37.9, 33.5, 31.1, 28.4, 26.6. HRMS (m/z): C32H38N5O8 [M+H]⁺ found: 620.2731. Calculated: 620.2720. [a]_D ²⁰ -126 (c 1.0, CHCh).

{2- [( LS.6.S.9.S)-6-/‘cr/‘- But y I- l-cyano-9- [(25)-2- hydro xy-3-methylbutanamido] -8-oxo- 19-oxa-4,7 - diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-3-yl]-l,3-oxazol-4-yl}methyl methyl carbonate (DZA-161). Compound was prepared according to General procedure C from macrocycle 41 (135 mg; 0.22 mmol), TFA (335 pL; 4.36 mmol; 20 equiv.) in DCM (1 mL) in 2 h and 5-HzVA (39 mg; 0.33 mmol; 1.5 equiv.), EDC^XHC1 (84 mg; 0.44 mmol; 2 equiv.), HOBt (88 mg; 0.66 mmol; 3 equiv.), DIPEA (189 pL; 1.09 mmol; 5 equiv.) in DMF (1 mL) in 1 h. Product DZA-161 was purified by reverse phase flash chromatography (10% to 50% MeCN in 0.01% TFA in water) to afford 65 mg (48%) as a white amorphous solid.

^XH NMR (400 MHz, MeOD) 5 8.16 (s, 1H), 7.37 - 7.30 (m, 2H), 6.98 (s, 1H), 5.20 (d, J= 15.1 Hz, 1H), 5.17 (d, J= 15.1 Hz, 1H), 4.75 - 4.72 (m, 1H), 4.55 (dd, J= 11.8, 3.8 Hz, 1H), 3.86 (d, J= 3.8 Hz, 1H), 3.79 (s, 3H), 3.28 - 3.05 (m, 3H), 3.00 - 2.83 (m, 3H), 2.09 (sept d, J= 6.8, 3.8 Hz, 1H), 1.01 (s, 9H), 1.00 (d, J= 6.8 Hz, 3H), 0.89 (d, J= 6.8 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.8, 173.9, 164.2, 157.0, 155.9, 149.8, 142.8, 142.5, 140.4, 138.5, 136.8, 131.8, 129.7, 128.2, 126.6, 119.5, 76.8, 61.9, 59.6, 56.3, 55.5, 47.7, 39.3, 39.2, 34.3, 33.2, 31.7, 26.8, 19.5, 16.3. HRMS (m/z): C₃2H38N₅O₈ [M+H]⁺ found: 620.2736. Calculated: 620.2720. [a]_D ²⁰ -143 (c 1.0, MeOH).

Ethyl (2£)-3-{2-[(15',65',95)-9-{[(tert-butoxy)carbonyl]amino}-6-tert-butyl-l-cyano-8-oxo-19- oxa-4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-3-yl]-l,3-oxazol-4- yl}prop-2-enoate (42). To a solution of alcohol 26 (380 mg; 0.68 mmol) in dry DCM (4 mL) DMP (316 mg; 0.74 mmol, 1.1 equiv.) was added in one portion. The resulting suspension was stirred at room temperature for 1 h. Then the suspension was diluted with EtOAc and washed with aqueous IM Na2S2C>3 and aqueous saturated NaHCCF. Organic layer was washed with brine, dried (Na2SO4), evaporated. The crude aldehyde (288 mg; 76%) was used in the next step without additional purification.

To a solution of triethyl phosphonoacetate (255 pL; 1.29 mmol, 2.5 equiv.) in THF (1 mL) was added sodium hydride (60% in mineral oil; 52 mg; 1.29 mmol, 2.5 equiv.) at 0 °C. The solution was stirred for 1 h at room temperature before being added to a solution of the crude aldehyde (288 mg; 0.52 mmol) in THF (1 mL) at 0 °C. The mixture was stirred at room temperature for 15 min. Then the reaction mixture was quenched with aqueous saturated NH4CI and extracted with EtOAc (x2). Organic layers were combined, washed with brine, dried (Na2SO4) and evaporated. The residue was purified with reverse phase column chromatography (from 10% to 50% MeCN in 0.01% TFA in water) to give ester 42 (115 mg; 36%) as a white amorphous solid.

^XH NMR (400 MHz, CDCh) 8 7.91 (s, 1H), 7.55 (d, J= 15.5 Hz, 1H), 7.25 - 7.22 (m, 2H), 7.13 (d, J= 1.1 Hz, 1H), 6.79 (d, = 15.5 Hz, 1H), 5.82 (d, = 7.7 Hz, 1H), 5.28 (d, = 9.2 Hz, 1H), 4.79 (d, J= 7.7 Hz, 1H), 4.26 (q, J= 7.1 Hz, 2H), 3.94 (ddd, J= 12.3, 9.2, 3.4 Hz, 1H), 3.35 - 3.14 (m, 3H), 3.01 - 2.84 (m, 3H), 1.43 (s, 9H), 1.34 (t, J= 7.1 Hz, 3H), 1.00 (s, 9H). ¹³C NMR (101 MHz, CDCh) 5 171.9, 166.9, 161.7, 155.4, 154.9, 148.5, 141.4, 140.8, 139.4, 138.8, 135.5, 131.8, 130.2, 129.5, 127.5, 125.5, 121.4, 118.2, 80.6, 60.7, 58.2, 58.0, 46.5, 38.6, 37.9, 33.6, 31.1, 28.4, 26.6, 14.5. HRMS (m/z): C34H40N5O7 [M+H]⁺ found: 630.2919. Calculated: 630.2928. [a]_D ²⁰ -117 (c 1.0, CHCh). Ethyl (2£)-3-{2-[( LS.6.S.9. )-6-/‘ r/‘-biityl-l-cy:ino-9-|(2.S)-2-hydroxy-3-niethylbiitanainido|-8- oxo-19-oxa-4,7-diazatetracyclo[9.5.2.1², .O¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-3-yl]-l,3- oxazol-4-yl}prop-2-enoate (DZA-148). Compound was prepared according to General procedure C from macrocycle 42 (100 mg; 0.16 mmol), TFA (122 pL; 1.59 mmol; 10 equiv.) in DCM (1 mL) in 1 h and 5-HzVA (28 mg; 0.24 mmol; 1.5 equiv.), EDC^XHC1 (61 mg; 0.32 mmol; 2 equiv.), HOBt (64 mg; 0.48 mmol; 3 equiv.), DIPEA (137 pL; 0.79 mmol; 5 equiv.) in DMF (2 mL) in 1 h. Product DZA-148 was purified by reverse phase flash chromatography (10% to 50% MeCN in 0.01% TFA in water) to afford 70 mg (70%) as a white amorphous solid.

‘HNMR (400 MHz, MeOD) 5 8.33 (d, J= 7.5 Hz, 1H), 8.33 (s, 1H, overlapping with 8.33 doublet), 7.63 (d, J = 15.6 Hz, 1H), 7.40 - 7.30 (m, 2H), 6.99 (s, 1H), 6.73 (d, J = 15.6 Hz, 1H), 4.76 - 4.67 (m, 1H), 4.56 (dd, J = 11.8, 3.8 Hz, 1H), 4.26 (q, J= 7.1 Hz, 2H), 3.87 (d, J = 3.8 Hz, 1H), 3.36 - 3.26 (m, 1H, overlapping with MeOD), 3.24 - 3.08 (m, 2H), 3.04 - 2.90 (m, 2H), 2.90 (dd, J= 12.4, 3.9 Hz, 1H), 2.09 (sept d, J= 6.9, 3.8 Hz, 1H), 1.34 (t, J = 7.1 Hz, 3H), 1.01 (s, 9H), 1.00 (d, J= 6.9 Hz, 3H), 0.90 (d, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.8, 174.0, 168.2, 164.1, 156.2, 150.3, 142.9, 142.4, 142.1, 139.7, 136.8, 133.4, 131.8, 129.8, 128.1, 126.6, 121.6, 119.4, 76.8, 61.8,

59.8, 56.3, 47.8, 39.5, 39.2, 34.4, 33.2, 31.8, 26.8, 19.5, 16.3, 14.6. HRMS (m/z): C34H40N5O7 [M+H]⁺ found: 630.2957. Calculated: 630.2928. [a]_D ²⁰ -148 (c 1.0, MeOH).

Ethyl 3- {2- [( 15.6.S'.9.S')-6-/cr/-bu ty 1- l-cyano-9- [(25)-2-hydroxy-3-methylbutanamido] -8-oxo- 19- oxa-4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-3-yl]-l,3-oxazol-4- yljpropanoate (DZA-149). To a solution of alkene (35 mg; 0.056 mmol) in dry MeOH (1 ML) was added Pd/C (10%; 6 mg; 0.006 mmol, 0.1 equiv.) in one portion at room temperature. Then H2 was barbotated through the suspension for 30 minutes. The resulting suspension was filtrated through celite and evaporated. The resulting yellowish amorphous solid was purified with reverse phase flash chromatography (10% to 50% MeCN in 0.01% TFA in water) to give ester DZA-149 (25 mg; 71%) as a white amorphous solid.

‘HNMR (400 MHz, MeOD) 5 8.34 (d, J= 7.5 Hz, 1H), 7.86 (s, 1H), 7.39 - 7.28 (m, 2H), 6.96 (d, J = 1.4 Hz, 1H), 4.74 - 4.69 (m, 1H), 4.55 (dd, J= 11.8, 3.8 Hz, 1H), 4.15 (q, J= 1A Hz, 2H), 3.86 (d, J= 3.8 Hz, 1H), 3.29 - 3.05 (m, 3H), 3.01 - 2.84 (m, 5H), 2.82 - 2.69 (m, 2H), 2.09 (sept d, J= 6.9, 3.8 Hz, 1H), 1.25 (t, J= 1A z, 3H), 1.00 (s, 9H), 1.00 (d, J= 6.9 Hz, 3H), 0.89 (d, J= 6.9 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.8, 174.3, 174.0, 164.1, 155.3, 149.4, 142.8, 142.7, 142.3, 137.4, 136.7, 131.8, 129.7, 128.5, 126.5, 119.5, 76.8, 61.7, 59.7, 56.3, 47.8, 39.4, 39.2, 34.3, 33.8, 33.2,

31.8, 26.8, 22.5, 19.5, 16.3, 14.5. HRMS (m/z): C34H42N5O7 [M+H]⁺ found: 632.3087. Calculated: 632.3084. [a]_D ²⁰ -149 (c 1.0, MeOH). The synthesis of diazonamide A analog DZA-176 is shown in Scheme 12.

Accordingly, methyl ester of 32 was hydrolyzed and coupled with (5)-(+)-2-amino-propan-l-ol to give amide 43. Primary alcohol of 43 was oxidized to aldehyde and after further oxazole cyclization reaction in the presence of PPha and 1,2-dibromotetrachloroethane under basic conditions was converted into methyl bioxazole 44. After N-Boc cleavage with TFAthe resulting amine was coupled with fS')-2-hydroxy-3 -methylbutanoic acid (5-HzVA; 7) to give analog DZA-176 (Scheme 12).

Scheme 12. Synthesis of diazonamide A analog DZA-176.

ZczZ- Butyl \-|( LS.6.S.9.S)-6-Zcz'Z-biityl-l-cyano-3-(4-methyl-l .3-oxazol-2-yl)-8-oxo-l 9-oxa-4.7- diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-9-yl]carbamate (44). Methyl ester 32 (385 mg; 0.74 mmol) was dissolved in THF (4 mL). Separately solid LiOHxEEO (93 mg; 0.2.21 mmol; 3 equiv.) was dissolved in water (2 mL) and added to the reaction mixture. Emulsion was stirred at room temperature for 30 min, then aqueous IN HC1 was added. The resulting mixture was extracted with EtOAc (*2), combined organic layers were washed with brine, dried (Na2SO4) and evaporated. The crude carboxylic acid was used in the next step without additional purification. The off-white amorphous solid was dissolved in anhydrous DMF (5 mL) and 2-amino-3,3,3- trifluoropropan-l-ol hydrochloride (183 mg; 1.11 mmol; 1.5 equiv.), EDC^XHC1 (212 mg; 1.11 mmol; 1.5 equiv.) and HOBt (199 mg; 1.47 mmol; 2 equiv.) were added followed by DIPEA (382 pL; 2.21 mmol; 3 equiv.). The solution was stirred at room temperature for 1 h. The solution was diluted with aqueous saturated NH4CI and EtOAc. The layers were separated and the organic phase was washed with brine, dried (Na2SO4) and evaporated. The resulting amide 43 was used in the next step without additional purification.

To a stirred solution of amide 43 (135 mg; 0.24 mmol) in DCM (1.5 mL) was added NaHCOa (40 mg; 0.48 mmol; 2 equiv.) followed by DMP (122 mg; 0.29 mmol; 1.2 equiv). The reaction mixture was stirred at room temperature for 1 h before being quenched with aqueous saturated NaHCCh and Na2S20a. The mixture was stirred vigorously for 1 h before being extracted with DCM (x2). The combined organic extracts were then dried (Na2SO4) and evaporated to afford a crude aldehyde, which was used without further purification. To a stirred solution of the crude aldehyde in DCM (2 mL) at 0°C was added sequentially DIPEA (165 pL, 0.96 mmol; 4 equiv.), PPha (125 mg, 0.96 mmol;

2 equiv.) and Br2C2C14 (155 mg, 0.48 mmol; 2 equiv.). The reaction mixture was allowed to warm to room temperature and stirred for 1 h before being recooled to 0°C and DBU (143 pL, 0.96 mmol; 4 equiv.) added. The reaction mixture was then stirred for 2 h at room temperature before being quenched with H2O and extracted with DCM (*2). The combined organic extracts were then dried (Na2SO4) and evaporated. The resulting orange oil was purified with direct phase flash chromatography (10% to 50% EtOAc in hexanes) to give bioxazole 44 (40 mg; 31%) as a white amorphous solid.

'H NMR (400 MHz, CDCh) 8 7.53 (q, J= 1.2 Hz, 1H), 7.25 - 7.19 (m, 2H), 7.12 (s, 1H), 5.67 (d, J = 7.9 Hz, 1H), 5.22 (d, J = 9.3 Hz, 1H), 4.79 (d, J= 7.9 Hz, 1H), 3.90 (ddd, J = 12.3, 9.3, 3.4 Hz, 1H), 3.34 - 3.07 (m, 3H), 3.01 - 2.79 (m, 3H), 2.27 (d, J= 1.3 Hz, 3H), 1.43 (s, 9H), 1.00 (s, 9H). ¹³C NMR (101 MHz, CDCh) 6 171.8, 161.6, 155.3, 153.9, 147.2, 141.3, 141.1, 138.4, 135.5, 135.3,

130.2, 129.5, 128.2, 125.4, 118.5, 80.5, 58.3, 57.9, 46.4, 38.7, 38.0, 33.5, 31.1, 28.4, 26.6, 11.9. HRMS (m/z): C30H36N5O5 [M+H]⁺ found: 546.2720. Calculated: 546.2716. [a]_D ²⁰ -125 (c 1.0, CHCh).

(2.S)-\-|( l.S.6.S.9.S)-6-/‘cr/‘-biityl-l-cyano-3-(4-methyl-1.3-oxazol-2-yl)-8-oxo-19-oxa-4.7- diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-9-yl]-2-hydroxy-3- methylbutanamide (DZA-176). Compound was prepared according to General procedure C from macrocycle 44 (40 mg; 0.073 mmol), TFA (56 pL; 0.73 mmol; 10 equiv.) in DCM (0.5 mL) in 2 h and 5-HzVA (13 mg; 0.11 mmol; 1.5 equiv.), EDC^XHC1 (42 mg; 0.22 mmol; 3 equiv.), HOBt (30 mg; 0.22 mmol; 3 equiv.), DIPEA (63 pL; 0.37 mmol; 5 equiv.) in DMF (0.5 mL) in 1 h. Product DZA-176 was purified by reverse phase flash chromatography (10% to 50% MeCN in 0.01% TFA in water) to afford 28 mg (70%) as a white amorphous solid.

’H NMR (400 MHz, MeOD) 5 8.34 (d, J= 7.5 Hz, 1H), 8.00 (d, J= 8.5 Hz, 1H), 7.82 (s, 1H), 7.42 - 7.26 (m, 2H), 6.94 (s, 1H), 4.71 (d, J= 7.5 Hz, 1H), 4.62 - 4.45 (m, 1H), 3.86 (d, J= 3.8 Hz, 1H), 3.26 (dd, J= 15.5, 7.7 Hz, 1H), 3.18 (dd, J= 12.1, 12.1 Hz, 1H), 3.15 - 3.07 (m, 1H), 3.00 - 2.92 (m, 1H), 2.91 - 2.82 (m, 2H), 2.27 (d, J= 1.3 Hz, 3H), 2.09 (sept d, J= 6.9, 3.8 Hz, 1H), 1.01 (d, J= 6.9 Hz, 3H), 1.00 (s, 9H), 0.89 (d, J= 6.9 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.8, 173.9, 164.1,

155.2, 149.3, 142.8, 142.7, 139.3, 137.3, 136.7, 131.8, 129.6, 128.5, 126.5, 119.5, 76.8, 59.7, 56.3, 47.7, 39.5, 39.2, 34.3, 33.2, 31.7, 26.8, 19.5, 16.3, 11.4. HRMS (m/z): C30H36N5O5 [M+H]⁺ found: 546.2725. Calculated: 546.2716. [a]_D ²⁰ -109 (c 1.0, MeOH). The synthesis of diazonamide A analogs DZA-156-159 is shown in Scheme 13.

Accordingly, hydroxamic acid DZA-156 and its derivatives DZA-157-159 were obtained from analog DZA-129 in two-step reaction sequence. After methyl ester hydrolysis the resulting acid was coupled with corresponding hy dr oxy/ alkoxy amine to give analogs DZA-156-159 (Scheme 13).

Scheme 13. Synthesis of diazonamide A analogs DZA-156-159

General procedure D (hydroxamic acid formation):

Methyl ester DZA-129 was dissolved in THF. Separately solid LiOHxH^O was dissolved in water and added to the reaction mixture. Emulsion was stirred at room temperature for 30 min, then aqueous HC1 (1 N) was added. The resulting mixture was extracted with EtOAc (*3), combined organic layers were washed with brine, dried (ISfeSCh) and evaporated. To the resulting crude acid was added the corresponding hydroxylamine and dissolved in dry DMF. Then HATU was added followed by DIPEA. The resulting solution was stirred at room temperature to full conversion (1 h). The solution was diluted with aqueous saturated NEUC1 and EtOAc. The layers were separated and the organic phase was washed with brine, dried (Na2SO4) and evaporated. Pure products were obtained after column chromatography.

2- [( l.S.6.S.9.S)-6-/‘cr/‘- But l- l-cyano-9- [(25)-2-hydroxy-3-methylbutanamido] -8-oxo- 19-oxa-4,7 - diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-3-yl]-7V-hydroxy-l,3-oxazole-4- carboxamide (DZA-156). Compound was prepared according to General procedure D from macrocycle DZA-129 (134 mg; 0.23 mmol), LiOHxEEO (51 mg; 0.68 mmol; 3 equiv.) in THF/water (1: 1 v/v; 1.5 mL) in 30 minutes and hydroxylamine ^XHC1 (79 mg; 1.14 mmol; 5 equiv.), HATU (112 mg; 0.30 mmol; 1.3 equiv.), DIPEA (275 pL; 1.59 mmol; 7 equiv.) in DMF (2 mL) in 1 h. Product DZA-156 was purified by reverse phase flash chromatography (10% to 50% MeCN in 0.01% TFA in water) to afford 42 mg (31%) as a white amorphous solid.

’H NMR (400 MHz, MeOD) 5 8.59 (s, 1H), 7.42 - 7.26 (m, 2H), 6.97 (s, 1H), 4.74 - 4.70 (m, 1H), 4.55 (dd, J= 11.8, 3.8 Hz, 1H), 3.86 (d, J= 3.8 Hz, 1H), 3.30 - 3.24 (m, 1H, overlapping with MeOD), 3.22 - 3.13 (m, 2H), 3.02 - 2.94 (m, 1H), 2.94 - 2.85 (m, 2H), 2.09 (sept d, J = 6.9, 3.8 Hz, 1H), 1.01 (s, 9H), 1.00 (d, J= 6.9 Hz, 3H), 0.89 (d, J= 6.9 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.8, 174.0, 164.4, 160.2, 155.7, 150.5, 143.6, 142.9, 142.5, 136.8, 131.9, 129.7, 127.8, 126.6, 119.5, 76.8, 59.7, 56.2, 47.8, 39.4, 39.1, 34.4, 33.2, 31.8, 26.7, 19.5, 16.3. HRMS (m/z): C30H35N6O7 [M+H]⁺ found: 591.2579. Calculated: 591.2567. [a]_D ²⁰ -147 (c 1.0, MeOH).

2- [( 15,6$,95)-6-terCButyl- l-cyano-9- [(25)-2-hydroxy-3-methylbutanamido] -8-oxo- 19-oxa-4,7 - diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-3-yl]-7V-hydroxy-7V-methyl-l,3- oxazole-4-carboxamide (DZA-157). Compound was prepared according to General procedure D from macrocycle DZA-129 (50 mg; 0.087 mmol), LiOHxH2O (11 mg; 0.26 mmol; 3 equiv.) in THF/water (1 : 1 v/v; 1.5 mL) in 30 minutes and 7V-methylhydroxylamine^xHCl (36 mg; 0.43 mmol; 5 equiv.), HATU (43 mg; 0.11 mmol; 1.3 equiv.), DIPEA (180 pL; 1.04 mmol; 12 equiv.) in DMF (1 mL) in 1 h. Product DZA-157 was purified by reverse phase flash chromatography (10% to 50% MeCN in 0.01% TFA in water) to afford 33 mg (63%) as a white amorphous solid.

^XH NMR (400 MHz, MeOD) 5 8.70 (s, 1H), 7.38 - 7.29 (m, 2H), 6.98 (s, 1H), 4.76 (s, 1H), 4.54 (dd, J = 11.8, 3.8 Hz, 1H), 3.86 (d, J= 3.8 Hz, 1H), 3.43 (s, 3H), 3.29 - 3.09 (m, 3H), 3.01 - 2.91 (m, 2H), 2.88 (dd, J= 12.5, 3.8 Hz, 1H), 2.09 (sept d, J= 6.9, 3.8 Hz, 1H), 1.01 (s, 9H), 1.00 (d, J= 6.8 Hz, 3H), 0.89 (d, J= 6.8 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.8, 174.0, 164.3, 162.2, 154.9,

150.3, 145.7, 142.8, 142.6, 136.8, 135.6, 131.8, 129.6, 128.0, 126.6, 119.4, 76.8, 59.6, 56.2, 47.8, 39.2, 37.1, 34.3, 33.2, 31.8, 26.8, 19.5, 16.3. HRMS (m/z): C31H37N6O7 [M+H]⁺ found: 605.2744. Calculated: 605.2724. [a]_D ²⁰ -161 (c 1.0, MeOH).

2- [( 15',65',95)-6-ferf-Butyl- l-cyano-9- [(25)-2-hydroxy-3-methylbutanamido] -8-oxo- 19-oxa-4,7 - diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-3-yl]-7V-methoxy-l,3-oxazole-4- carboxamide (DZA-158). Compound was prepared according to General procedure D from macrocycle DZA-129 (134 mg; 0.23 mmol), LiOHxH2O (51 mg; 0.68 mmol; 3 equiv.) in THF/water (1: 1 v/v; 1.5 mL) in 30 minutes and O-methylhydroxylamine^xHCl (36 mg; 0.43; 5 equiv.), HATU (112 mg; 0.30 mmol; 1.3 equiv.), DIPEA (180 pL; 1.04 mmol; 12 equiv.) in DMF (2 mL) in 1 h. Product DZA-158 was purified by reverse phase flash chromatography (10% to 50% MeCN in 0.01% TFA in water) to afford 42 mg (31%) as a white amorphous solid.

^XH NMR (400 MHz, MeOD) 5 8.64 (s, 1H), 7.39 - 7.28 (m, 2H), 6.97 (s, 1H), 4.73 (s, 1H), 4.55 (dd, J = 11.8, 3.8 Hz, 1H), 3.86 (d, J = 3.7 Hz, 1H), 3.84 (s, 3H), 3.30 - 3.24 (m, 1H), 3.24 - 3.12 (m, 2H), 3.01 - 2.83 (m, 3H), 2.09 (sept d, J= 6.9, 3.8 Hz, 1H), 1.01 (s, 9H), 1.00 (d, J= 6.9 Hz, 3H), 0.89 (d, J= 6.9 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.8, 174.0, 164.4, 160.0, 155.8, 150.6,

144.3, 142.8, 142.5, 136.8, 136.7, 131.9, 129.7, 127.8, 126.6, 119.5, 76.8, 64.7, 59.7, 56.2, 47.8, 39.3, 39.1, 34.3, 33.2, 31.8, 26.7, 19.5, 16.3. HRMS (m/z): C31H37N6O7 [M+H]⁺ found: 605.2737.

Calculated: 605.2724. [a]_D ²⁰ -137 (c 1.0, MeOH).

2- [( LS.6.S.9.S)-6-/‘cr/‘- Butyl- l-cyano-9- 1 ( 2.S')-2-hy dro xy-3-met hy Ibu t a na in ido | -8-oxo- 19-oxa-4,7 - diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-3-yl]-7V-methoxy-7V-methyl-l,3- oxazole-4-carboxamide (DZA-159). Compound was prepared according to General procedure D from macrocycle DZA-129 (50 mg; 0.087 mmol), LiOHxlHbO (11 mg; 0.26 mmol; 3 equiv.) in THF/water (1 : 1 v/v; 1.5 mL) in 30 minutes and 7V,( -dimethylhydroxylamine (43 mg; 0.43 mmol; 5 equiv.), HATU (43 mg; 0.11 mmol; 1.3 equiv.), DIPEA (105 pL; 0.61 mmol; 7 equiv.) in DMF (1 mL) in 1 h. Product DZA-159 was purified by reverse phase flash chromatography (10% to 50% MeCN in 0.01% TFA in water) to afford 36 mg (67%) as a white amorphous solid.

‘H NMR (400 MHz, MeOD) 5 8.70 (s, 1H), 7.39 - 7.25 (m, 2H), 6.97 (s, 1H), 4.74 (s, 1H), 4.55 (dd, J = 11.8, 3.8 Hz, 1H), 3.87 (d, J= 3.8 Hz, 1H), 3.86 (s, 3H), 3.45 (s, 3H), 3.29 - 3.22 (m, 1H), 3.22 - 3.09 (m, 2H), 3.04 - 2.92 (m, 2H), 2.89 (dd, J = 12.4, 3.8 Hz, 1H), 2.09 (sept d, J= 6.9, 3.8 Hz, 1H), 1.01 (s, 9H), 1.00 (d, 7= 6.8 Hz, 3H), 0.89 (d, 7= 6.8 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.8, 173.9, 164.3, 162.1, 155.3, 150.4, 146.0, 142.9, 142.5, 136.8, 135.5, 131.9, 129.6, 127.9, 126.6, 119.4, 76.8, 62.1, 59.6, 56.3, 55.1, 47.8, 39.2, 39.2, 34.3, 33.2, 31.8, 26.8, 19.5, 16.3. HRMS (m/z): C32H39N6O7 [M+H]⁺ found: 619.2890. Calculated: 619.2880. [a]_D ²⁰ -140 (c 1.0, MeOH).

The synthesis of diazonamide A analogs DZA-132-134 and DZA-138 is shown in Scheme 14.

1,2,4-Oxadiazole-containing analogs DZA-132-134 were prepared from analog DZA-129 in one step in reaction with corresponding hydroxyamidine in the presence of K2CO3. Accordingly, analog DZA- 138 was obtained after methyl ester hydrolysis in the presence of LiOHxH2O (Scheme 14).

Scheme 14. Synthesis of diazonamide A analogs DZA-132-134 and DZA-138 General procedure E (1,2,4-oxadiazole formation):

Methyl ester DZA-129 was dissolved in dry toluene and K2CO3, A-hydroxyamidine were added to the reaction mixture. The resulting suspension was stirred at 110°C to full conversion (4-10 h). The solution was diluted with aqueous saturated NH4CI and EtOAc. The layers were separated and the organic phase was washed with brine, dried (Na2SO4) and evaporated. Pure products were obtained after column chromatography.

(2S)-N- [( 15',65',95)-6-ferf-Butyl- l-cyano-3- [4-(3-methyl- 1 ,2,4-oxadiazol-5-yl)- 1 ,3-oxazol-2-yl] - 8-oxo-19-oxa-4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-9-yl]-2- hydroxy-3-methylbutanamide (DZA-132). Compound was prepared according to General procedure E from macrocycle DZA-129 (60 mg; 0.10 mmol), K2CO3 (42 mg; 0.31 mmol; 3 equiv.) and A-hydroxyacetamidine (38 mg; 0.51 mmol; 5 equiv.) in toluene (1 mL) in 4 h. Product DZA-132 was purified by reverse phase flash chromatography (10% to 70% MeCN in water) to afford 24 mg (38%) as a white amorphous solid.

‘H NMR (400 MHz, MeOD) 5 8.99 (s, 1H), 7.39 - 7.31 (m, 2H), 7.02 (s, 1H), 4.76 (s, 1H), 4.56 (dd, J= 11.8, 3.8 Hz, 1H), 3.87 (d, J= 3.8 Hz, 1H), 3.36 - 3.25 (m, 1H, overlapping with MeOD), 3.24 - 3.11 (m, 2H), 3.06 - 2.94 (m, 2H), 2.90 (dd, J= 12.4, 3.8 Hz, 1H), 2.47 (s, 3H), 2.09 (sept d, J= 6.9, 3.8 Hz, 1H), 1.02 (s, 9H), 1.01 (d, J = 6.9 Hz, 3H), 0.90 (d, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.8, 174.0, 170.3, 169.1, 164.3, 157.1, 150.9, 144.6, 142.9, 142.3, 136.8, 131.9, 130.3, 129.7, 127.6, 126.6, 119.4, 76.8, 59.6, 56.3, 47.8, 39.19, 39.15, 34.4, 33.2, 31.8, 26.8, 19.5, 16.3, 11.3. HRMS (m/z): C32H36N7O6 [M+H]⁺ found: 614.2766. Calculated: 614.2727. [a]_D ²⁰ -164 (c 1.0, MeOH).

(25)-A-[(15',65',95)-3-[4-(3-Benzyl-l,2,4-oxadiazol-5-yl)-l,3-oxazol-2-yl]-6-ferf-butyl-l-cyano-8- oxo-19-oxa-4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-9-yl]-2- hydroxy-3-methylbutanamide (DZA-133). Compound was prepared according to General procedure E from macrocycle DZA-129 (55 mg; 0.093 mmol), K2CO3 (39 mg; 0.28 mmol; 3 equiv.) and A-hydroxy-2-phenylethanimidamide (70 mg; 0.47 mmol; 5 equiv.) in toluene (1 mL) in 10 h. Product DZA-133 was purified by reverse phase flash chromatography (10% to 70% MeCN in water) to afford 38 mg (59%) as a white amorphous solid.

’H NMR (400 MHz, DMSO-t ,) 8 9.38 (s, 1H), 8.40 (d, J= 7.9 Hz, 1H), 7.62 (d, J= 8.4 Hz, 1H), 7.38 - 7.34 (m, 4H), 7.32 - 7.29 (m, 2H), 7.30 - 7.25 (m, 1H), 6.99 (s, 1H), 5.42 (d, J= 6.1 Hz, 1H), 4.74 (d, J= 7.8 Hz, 1H), 4.52 (ddd, J= 11.9, 8.4, 3.9 Hz, 1H), 4.21 (s, 2H), 3.72 (dd, J= 6.1, 3.9 Hz, 1H), 3.26 - 3.14 (m, 1H), 3.10 - 2.96 (m, 3H), 2.93 - 2.76 (m, 2H), 1.99 (sept d, J= 6.8, 3.8 Hz, 1H), 0.96 (s, 9H), 0.90 (d, J = 6.8 Hz, 4H), 0.79 (d, J = 6.8 Hz, 4H). ¹³C NMR (101 MHz, DMSO-t ,) 6 172.4, 171.8, 170.0, 168.7, 162.9, 155.0, 148.3, 144.5, 140.9, 140.2, 135.6, 135.5, 130.7, 129.0, 128.7,

128.2, 128.1, 127.0, 125.3, 125.3, 118.6, 74.8, 57.0, 54.3, 45.7, 38.1, 36.5, 33.1, 31.5, 31.3, 30.5,

26.2, 19.1, 16.2. HRMS (m/z): C38H40N7O6 [M+H]⁺ found: 690.3021. Calculated: 690.3040. [a]_D ²⁰ - 126 (c 1.0, MeOH).

(25)-N-[(15',65',95)-6-terCButyl-l-cyano-8-oxo-3-[4-(3-phenyl-l,2,4-oxadiazol-5-yl)-l,3-oxazol- 2-yl]-19-oxa-4,7-diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-9-yl]-2- hydroxy-3-methylbutanamide (DZA-134). Compound was prepared according to General procedure E from macrocycle DZA-129 (60 mg; 0.10 mmol), K2CO3 (42 mg; 0.31 mmol; 3 equiv.) and benzamidoxime (69 mg; 0.51 mmol; 5 equiv.) in toluene (1 mL) in 10 h. Product DZA-134 was purified by reverse phase flash chromatography (10% to 70% MeCN in water) to afford 38 mg (55%) as a white amorphous solid.

‘HNMR (400 MHz, DMSO-tL) 8 9.50 (s, 1H), 8.42 (d, J= 7.9 Hz, 1H), 8.16 - 8.02 (m, 2H), 7.70 - 7.55 (m, 4H), 7.39 - 7.24 (m, 2H), 7.01 (s, 1H), 5.43 (d, J= 6.1 Hz, 1H), 4.76 (d, J= 7.8 Hz, 1H), 4.54 (ddd, J= 11.9, 8.4, 3.9 Hz, 1H), 3.73 (dd, J= 6.1, 3.9 Hz, 1H), 3.28 - 3.17 (m, 1H), 3.13 - 3.06 (m, 2H), 3.02 (dd, J= 11.9, 11.9 Hz, 1H), 2.96 - 2.85 (m, 1H), 2.82 (dd, J = 12.3, 3.9 Hz, 1H), 2.06 - 1.92 (m, 1H), 0.97 (s, 9H), 0.91 (d, J= 6.9 Hz, 3H), 0.80 (d, J= 6.9 Hz, 3H). ¹³C NMR (101 MHz, DMSO-t/e) 6 172.5, 171.8, 169.1, 168.3, 162.9, 155.2, 148.4, 144.7, 140.9, 140.2, 135.5, 131.9, 130.7,

129.4, 128.18, 128.16, 127.2, 125.8, 125.4, 125.3, 118.7, 74.9, 57.1, 54.3, 45.8, 38.1, 36.6, 33.1, 31.5, 30.5, 26.2, 19.1, 16.2. HRMS (m/z): C37H38N7O6 [M+H]⁺ found: 676.2897. Calculated: 676.2884. [a]_D ²⁰ -121 (c 1.0, MeOH).

2- [( 15',65',95)-6-terCButyl- l-cyano-9- [(25)-2-hydroxy-3-methylbutanamido] -8-oxo- 19-oxa-4,7 - diazatetracyclo[9.5.2.1², .0¹⁴,¹ ]nonadeca-2,4,ll,13,17-pentaen-3-yl]-l,3-oxazole-4-carboxylic acid (DZA-138). Methyl ester DZA-129 (33 mg; 0.056 mmol) was dissolved in THF (0.5 mL). Separately solid LiOH*H2O (7 mg; 0.17 mmol; 3 equiv.) was dissolved in water (0.2 mL) and added to the reaction mixture. Emulsion was stirred at room temperature for 30 min, then aqueous IN HC1 was added. The resulting mixture was extracted with EtOAc (x2), combined organic layers were washed with brine, dried (Na2SO4) and evaporated. The resulting white amorphous solid was purified with reverse phase flash chromatography (10% to 50% MeCN in 0.01% TFA in water) to give carboxylic acid DZA-138 (22 mg; 68%) as a white amorphous solid.

‘H NMR (400 MHz, MeOD) 5 8.71 (s, 1H), 7.43 - 7.25 (m, 2H), 6.98 (s, 1H), 4.75 (s, 1H), 4.54 (dd, J= 11.8, 3.8 Hz, 1H), 3.86 (d, = 3.8 Hz, 1H), 3.29 - 3.10 (m, 3H), 3.02 - 2.92 (m, 2H), 2.88 (dd, J = 12.4, 3.8 Hz, 1H), 2.08 (sept d, J= 13.4, 6.9, 3.8 Hz, 1H), 1.01 (s, 9H), 1.00 (d, J= 6.9 Hz, 3H), 0.89 (d, J= 6.9 Hz, 3H). ¹³C NMR (101 MHz, MeOD) 5 175.8, 174.0, 164.4, 163.6, 156.2, 150.5, 146.7, 142.9, 142.5, 136.8, 136.0, 131.9, 129.6, 127.8, 126.6, 119.4, 76.8, 59.6, 56.2, 47.8, 39.18, 39.15, 34.3, 33.2, 31.8, 26.8, 19.5, 16.3. HRMS (m/z): C30H34N5O7 [M+H]⁺ found: 576.2470. Calculated: 576.2458. [a]_D ²⁰ -135 (c 1.0, MeOH).

Antiproliferative activity in vitro

Anticancer activity of all synthesized compounds above was tested in vitro using cytotoxicity assay. Thus, monolayer tumor cell lines A2058 (metastatic melanoma), U937 (myeloid leukemia), MDA- MB-231 (breast adenocarcinoma), MDA-MB-435 (metastatic melanoma) and non-cancer HEK-293 (human embryonic kidney cells) were cultured in standard medium DMEM (Dulbecco¹ s modified Eagle's medium) ("Sigma") supplemented with 10% fetal bovine serum ("Sigma"). About 2-9 10⁴ cells/mL (depending on line nature) were placed in 96-well plates immediately after compounds were added to the wells. Untreated cells were used as a control. The plates were incubated for 72 h, 37 °C, 5% CO2. The number of surviving cells was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolinium bromide (MTT). MTT-test: after incubating culture medium was removed and 200 pL fresh medium with 10 mM HEPES was added in each well of the plate, than 20 pL MTT (2mg/mL in HBSS) was added. After incubation (3 hr, 37°C, 5% CO2), the medium with MTT was removed and 200 pL DMSO were added at once to each sample. The samples were tested at 540 nm on Anthos HT II photometer. The results of cell culture-based studies are summarized in Table 2.

All compounds were tested on A2058 (metastatic melanoma) cell line. Then compounds with highest cytotoxicity against this line were further tested on U937 (myeloid leukemia), MDA-MB-231 (breast adenocarcinoma), MDA-MB-435 (metastatic melanoma) and non-cancer HEK-293 (human embryonic kidney cells). In general, tested compounds showed medium or low cytotoxicity against tumor cells. In partucular the series of compounds were most cytotoxic against melanoma cell lines. Notably, almost all derivatives are low toxic to normal HEK-293 cells showing high selectivity (> 10-fold). Compounds containing ester moiety at the oxazole have lower IC50 compared to those with different functional groups. DZA-129 has the lowest IC50 values against all cancer cells. However, the compound also has low selectivity against normal cell line. Compound DZA-174 in particular has highest selectivity against cancer cells compared to normal cell line indicating potential for future preclinical studies.

It should be also emphasized that DZA-129, a representative of indane-containing series exerts one to two orders of magnitude higher cytotoxicity against A2058, U937, and MDA-MB-231 cancer cell lines (see Table 2, line 2, below) as compared to the known oxindole subunit-containing analog DZA- 60 [8,9]:

Overall the selectivity against melanoma cancer cell lines makes the indane-containing compound series very promising as anticancer agents.

able 2. Cell viability data. In vitro cytotoxicity of diazonamide analogs on human tumor cell lines: A2058 (metastatic melanoma), U937 (myeloideukemia), MDA-MB-231 (breast adenocarcinoma), MDA-MB-435 (metastatic melanoma) and non-cancer HEK-293 (human embryonic kidney cells)

Cytotoxicity IC50, nM

# Compound # A2058 U937 MDA-MB-231 MDA-MB-435 HEK-293

1 Vinorelbine 2.41±0.21 4.21±0.61 4.50±0.42 n/a 48.0±11.3

2 DZA-129 12.9±1.9 1.93±0.7 130.6±20.7 40.09±6.0 190.9±24.3

3 DZA-132 186±16 157±2 n/a n/a >1000

4 DZA-133 687±79 729±75 n/a n/a >1000

5 DZA-134 156±18 110±8 n/a n/a >1000

6 DZA-135 20.4±1.3 22.2±1.2 n/a n/a >1000

7 DZA-137 167.4±16.5 64.94±9.3 >1000 299.4±59.9 >1000

8 DZA-138 >1000 >1000 >1000 >1000 >1000

9 DZA-139 12.37±1.5 9.71±2.1 290.2±42.5 n/a 324.9±79.0

10 DZA-140 >1000 261±65.9 >1000 n/a >1000

11 DZA-141 958.7±130.4 214±47 >1000 n/a >1000

12 DZA-142 643.4±91.7 692.3±20.2 >1000 n/a >1000

13 DZA-143 68.19±10.8 22.45±4.6 n/a n/a 540.4±66.7

14 DZA-144 574.2±76.1 100.8±22.6 n/a n/a >1000

15 DZA-145 >1000 >1000 n/a n/a >1000

16 DZA-146 >1000 177.9±40.6 >1000 n/a n/a

17 DZA-147 792.5±223.3 40.86±10.3 >1000 n/a n/a

18 DZA-148 541.0±182.1 94.7±17.5 >1000 n/a n/a

19 DZA-149 202.7±53.0 136.3±45.0 >1000 n/a n/a

20 DZA-150 >1000 n/a n/a n/a n/a

21 DZA-151 >1000 175 >1000 n/a n/a

22 DZA-152 >1000 n/a n/a n/a n/a

23 DZA-153 >1000 n/a n/a n/a n/a

24 DZA-154 >1000 n/a >1000 n/a >1000

25 DZA-156 >1000 n/a >1000 n/a >1000

26 DZA-157 212.3±54.6 n/a >1000 n/a >1000

27 DZA-158 864.6±190.8 n/a >1000 n/a >1000

28 DZA-159 220.9±51.6 n/a >1000 n/a >1000

29 DZA-160 319.4±105.2 n/a >1000 n/a >1000

30 DZA-161 60.1±8.9 n/a 432.9±54.5 62.05±8.9 >1000

31 DZA-162 >1000 n/a >1000 n/a n/a

32 DZA-163 100.3±10.0 n/a 736.2±176.1 n/a n/a

33 DZA-164 >1000 n/a >1000 n/a n/a

34 DZA-165 >1000 n/a >1000 n/a n/a

35 DZA-168 235.3±35.3 n/a 526.7±57.2 130.5±19.2 >1000

36 DZA-174 90.78±16.52 n/a n/a 80.8±10.88 >1000

37 DZA-175 480.1±86.7 n/a n/a 402.9±85.9 902.7±188.6

38 DZA-176 328.8±104.0 n/a n/a 334.8±36.8 591.8±135.0

Tubulin polymerization assay

An in vitro tubulin polymerization assay was performed to evaluate the effect of the series on microtubule dynamics. In the assay, enhancer (paclitaxel) control self-polymerization of soluble a/p- tubulin dimer to insoluble oligomers under buffered conditions is monitored by measuring changes in light scattering at 340 nm. A slope was calculated for the linear growth phase of the tubulin polymerization curve to render a comparison across the series more convenient (see Table 3). Enhancer control tubulin self-polymerization was measured in the presence of the previously synthesized analogs DZA.¹ To verify the correlation between antiproliferative activity of the macrocycles and effect they exert on tubulin polymerization dynamics, poorly cytotoxic macrocycles and analogs with the highest antiproliferative activities were examined. Finally, vinorelbine was also tested as the assay positive control (see Figure 1 enhancer control tubulin self-polymerization assay results for macrocycles DZA-129, DZA-137, DZA-138, DZA-141, DZA-145, DZA-147, DZA-156, DZA-161, DZA- 163, DZA-165, DZA-168, DZA-174, DZA-175 and vinorelbine (VNB) as a positive conrol of the assay).

Table 3. Tubulin polymerization assay results.

Entry Ligand" R² fit

1 Control 1.000 0.999

2 Vinorelbine (VNB) 0.010 0.999

3 DZA-129 0.021 0.999

4 DZA-137 0.061 0.998

5 DZA-138 0.019 0.999

6 DZA-141 0.058 0.998

7 DZA-145 0.551 0.999

8 DZA-147 0.075 0.999

9 DZA-156 0.040 0.999

10 DZA-161 0.040 0.999

11 DZA-163 0.033 0.999

12 DZA-165 0.0198 0.999

13 DZA-168 0.1979 0.999

15 DZA-175 0.0459 0.979

“ 2: 1 tubulin: ligand ratio; ^b Slope values are relative to that of control (entry 1)

¹ The assay was conducted following Enhancer Control Polymerization Assay Method from Tubulin Polymerization

Assay Kit manual by Cytoskeleton, Inc. (https://www.cytoskeleton.com/pdf-storage/datasheets/bk004p.pdf) Plasma stability assay

Stock solutions (100 pM) of test compound, propantheline bromide (positive control) and verapamil (negative control) are prepared in dimethyl sulfoxide (DMSO).

The assay procedure is performed in microcentrifuge tubes (1.5 mL). Mouse plasma with heparin (Innovative Research, Inc., Cat # IGMSCD1PLANAH50ML-36650, 495 pL in each tube) was preincubated at 37 °C for 10 minutes. Afterwards, 5 pL of stock solution (100 pM) was added. The spiked plasma samples containing 1 pM of a compound and 1 % of DMSO were incubated at 37 °C for 2 hours. Aliqouts of 20 pL were collected at 0, 15, 30, 60 and 120 min and added to 180 pL of 3: 1 acetonitrile: methanol, containing reserpine as an internal standard. The samples were centrifuged at 10000 rpm for 10 min and 150 pL of supernatant was diluted with 300 pL of 0.1 % formic acid for LC-MS/MS analysis.

The samples were analyzed by LC-MS/MS according to standard procedures on the Waters XevoTQ-Smicro system. Mass spectrometric settings were optimized for each of test compound. Chromatographic separation was performed on C18 BEH 1.7pm column (2><50mm).

The calculated peak area response ratio (peak area corresponding to test compound divided by that of an analytical internal standard) was normalized (% of response at Oh) and plotted against the incubation time.

Table 4. Plasma stability data

% of a compound found

Cell cycle studies. MDA-MB-435 cells were seeded in 24-well plates at a density of 1 x 10⁵ cells/mL. The cells were then allowed to rest and adhere to the plate for 5 hours. Subsequently, cells were treated with equitoxic concentrations (IC90) of vinorelbine and DZA-174 for 4, 8 and 24 hours.

Flow cytometry analysis of cell cycle progression of MDA-MB-435 cells after incubation with DZA- 174 (200 nM) for up to 24 hours. Quantitative results of the effects of DZA-174 on cell distribution among phases of the cell cycle in comparison with equitoxic concentration (30 nM) of vinorelbine (bottom) (Fig.2). The data are presented as the mean ± SEM of three independent experiments in triplicate.

To assess the distribution of cells between cell cycle phases, we performed cell cycle analysis using quantification of DNA content with the commercially available kit (Abeam, ab 139418) according to the manufacturer’s instructions. In brief, after incubation with the test compounds, cells were harvested using trypsin, washed with PBS, and fixed with 66% ice-cold ethanol. After fixation, cells were washed with PBS, and incubated with propidium iodide (PI) (50 pg/mL) and RNase (550 U/mL) solution for 30 min in the dark at 37 ° C. After incubation, the cells were transferred to ice and prepared for flow cytometry analysis using the BD FACS Melody Cell Sorter (BD Biosciences, San Jose, CA, USA). Quantitative results are expressed as the mean ± SEM. All experiments were carried out in triplicate with three technical replicates.

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Claims

Claims

1. A compound of general formula (I)

wherein

R¹ represents optional substituent at quaternary carbon;

R² represents optional substituent at oxazole;

R³ represents optional substituent at aromatic subunit;

Q represents C1-2 alkylene group or C1-2 heteroalkylene group; optical isomers and stereoisomers, mixtures thereof, including racemate, one or more enantiomeric forms, one or more diastereomeric forms, or mixtures thereof; pharmaceutically acceptable salts, hydrates, solvates and polymorphs thereof.

2. The compound according to Claim 1 wherein

R¹ is -CN, -C(=O)R⁴, -C(=O)OR⁴, -C(=O)N(R⁴)R⁵, -CH₂N(R⁴)R⁵, wherein:

R⁴, R⁵, independently represent, on each occasion when used herein, H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, arylCi-ealkyl, heteroaryl or heteroarylCi-ealkyl,

R⁴ and R⁵ taken together represent -V^A-W^A-X^A-Y^A-Z^A-, or -V^A-W^A-X^A-Y^A-, -V^A- W^A-X^A-, -V^A-W^A- wherein:

V^A represents, oxygen, sulfur, -NR⁶, -CR⁶R⁷

W^A represents oxygen, sulfur, -NR⁶, -CR⁶R⁷

X^A represents oxygen, sulfur, -NR⁶, -CR⁶R⁷ Y^A represents oxygen, sulfur, -NR⁶, -CR⁶R⁷

Z^A represents oxygen, sulfur, -NR⁶, -CR⁶R⁷, wherein:

R⁶, R⁷, independently represent, on each occasion when used herein, H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, arylCi-ealkyl, heteroaryl or heteroarylCi-ealkyl.

3. The compound according to Claim 1 wherein

R² is -F, -Cl, -Br, -I, -L-CF3, -L-CHF₂, -L-CFFF. -L-F, -L-OR⁸, -L-NR⁸R⁹, -L- C(=O)R⁸, -L-C(=N-OR⁸)R⁹, -L-C(=O)OR⁸, -L-C(=O)OCR⁸R⁹OC(=O)-L-R¹⁰, -L- C(=O)NR⁸R⁹, -L-C(=O)NR⁸-OR⁹, -L-CN, -L-NO2, -L-aryl, -L-arylCi-ealkyl, -L- heteroaryl; wherein heteroaryl is selected from:

wherein:

R^H represent H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, arylCi-ealkyl, heteroaryl, heteroarylCi-ealkyl,

R⁸, R⁹and R¹⁰ independently represent, on each occasion when used herein, H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, arylCi-ealkyl, heteroaryl, heteroarylCi-ealkyl

R⁸ and R⁹ taken together represent -V^B-W^B-X^B-Y^B-Z^B-, or -V^B-W^B-X^B-Y^B-, -V^B-

W^B-X^B-, -V^B-W^B- wherein:

V^B represents, oxygen, sulfur, -NR¹¹, -CRⁿR¹²

W^B represents oxygen, sulfur, -NR¹¹, -CRⁿR¹²

X^B represents oxygen, sulfur, -NR¹¹, -CRⁿR¹²

Y^B represents oxygen, sulfur, -NR¹¹, -CRⁿR¹²

Z^B represents oxygen, sulfur, -NR¹¹, -CRⁿR¹² wherein:

R¹¹, R¹², independently represent, on each occasion when used herein, H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, arylCi-ealkyl, heteroaryl, heteroarylCi-ealkyl

L represents -W^c-X^c-Y^c-, or -W^c-X^c-, or -W^c- wherein:

W^c is absent or represents oxygen, sulfur, -NR¹³, =CR¹³, -CR¹³R¹⁴

X^c represents oxygen, sulfur, -NR¹³, =CR¹³, -CR¹³R¹⁴

Y^c represents oxygen, sulfur, -NR¹³, =CR¹³, -CR¹³R¹⁴ wherein:

R¹³, R¹⁴ independently represent, on each occasion when used herein, H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, arylCi-ealkyl, heteroaryl, heteroarylCi-ealkyl

R¹³ and R¹⁴ taken together represent -V^D-W^D-X^D-Y^D-Z^D-, or -V^D-W^D-X^D-Y^D-, - _V ^D-W^D-X^D-, -V^D-W^D- wherein:

V^D represents, oxygen, sulfur, -NR¹⁵, -CR¹⁵R¹⁶

W^D represents oxygen, sulfur, -NR¹⁵, -CR¹⁵R¹⁶

X^D represents oxygen, sulfur, -NR¹⁵, -CR¹⁵R¹⁶

Y^D represents oxygen, sulfur, -NR¹⁵, -CR¹⁵R¹⁶

Z^D represents oxygen, sulfur, -NR¹⁵, -CR¹⁵R¹⁶ wherein:

R¹⁵, R¹⁶ independently represent, on each occasion when used herein, H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, arylCi-ealkyl, heteroaryl or heteroarylCi-ealkyl.

4. The compound according to Claim 1 wherein

R³ is -H, -F, -Cl, -Br, -I or Ci-ealkyl.

5. The compound according to Claim 1 wherein

Q represents -W^E-X^E-, or -W^E- wherein: W^E represents oxygen, sulfur, -S(=O)-, -SO2-, -CH2-;

X^E represents oxygen, sulfur, -S(=O)-, -SO2- or -CH2-.

6. The compound of formula (I) according to Claims 1-5 for the use in the treatment of tumors.

7. The compound of formula (I) according to Claims 6 for the use in the treatment of melanoma.

8. A pharmaceutical composition comprising compound according to Claims 1-5 and one or more pharmaceutically acceptable diluents, carriers or excipients.