Improved Total Synthesis of Tubulysins and Design, Synthesis, and Biological Evaluation of New Tubulysins with Highly Potent Cytotoxicities against Cancer Cells as Potential Payloads for Antibody-Drug Conjugates

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Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX −XXX

Improved Total Synthesis of Tubulysins and Design, Synthesis, and

Biological Evaluation of New Tubulysins with Highly Potent

Cytotoxicities against Cancer Cells as Potential Payloads for

Antibody−Drug Conjugates

K. C. Nicolaou,*^,†Rohan D. Erande,^†Jun Yin,^†Dionisios Vourloumis,^†,‡Monette Aujay,^#

Joseph Sandoval,^#Stefan Munneke,^#and Julia Gavrilyuk^#

^†Department of Chemistry, BioScience Research Collaborative, Rice University, 6100 Main Street, Houston, Texas 77005, United

States

^‡Laboratory of Chemical Biology of Natural Products & Designed Molecules, N.C.S.R “Demokritos”, 153 10 Agia Paraskevi, Athens,

Greece

^#AbbVie Stemcentrx, LLC, 450 East Jamie Court, South San Francisco, California 94080, United States

S

* Supporting Information

ABSTRACT: Improved, streamlined total syntheses of

natural tubulysins such as V (Tb45) and U (Tb46) and

pretubulysin D (PTb-D43), and their application to the

synthesis of designed tubulysin analogues (Tb44, PTb-D42,

PTb-D47−PTb-D49, and Tb50−Tb120), are described.

Cytotoxicity evaluation of the synthesized compounds against

certain cancer cell lines revealed a number of novel analogues

with exceptional potencies [e.g., Tb111: IC₅₀= 40 pM against

MES SA (uterine sarcoma) cell line; IC₅₀= 6 pM against HEK

293T (human embryonic kidney cancer) cell line; and IC₅₀

=

1.54 nM against MES SA DX (MES SA with marked multidrug

resistance) cell line]. These studies led to a set of valuable

structure−activity relationships that provide guidance to

further molecular design, synthesis, and biological evaluation studies. The extremely potent cytotoxic compounds discovered

in these investigations are highly desirable as potential payloads for antibody−drug conjugates and other drug delivery systems

for personalized targeted cancer chemotherapies.

evaluation.¹¹In this Article we report on a more extensive

study that included (a) streamlined total syntheses of the

1. INTRODUCTION

The tubulysins are among the most potent cytotoxic

natural tubulysins V (Tb45, Figure 1) and U (Tb46, Figure 1)

compounds ever discovered from Nature.¹⁻³Their mechanism

and pretubulysin D (PTb-D43, Figure 1); (b) design and

of action involves depolymerization of microtubules with

synthesis of numerous novel tubulysin analogues (i.e., PTb-

disintegration of the cytoskeleton as a consequence.⁴⁻⁶Isolated

D42, Tb44, PTb-D47−PTb-D49, and Tb50−Tb120, Figure

from the myxobacteria Archangium gephyra and Angiococcus

2); and (c) biological evaluation of the synthesized compounds.

disciformis,^7,8these natural products elicited intense research

eﬀorts directed toward their total synthesis, analogue design

and synthesis, and biological investigations as part of anticancer

These investigations led to the discovery of a number of

exceptionally potent antitumor agents particularly suited as

payloads for antibody−drug conjugates (ADCs)^50,51and other

drug discovery and development programs.⁹⁻¹⁴Thus, total

syntheses of the naturally occurring tubulysins A,¹⁵B,¹⁵C,¹⁶

D,¹⁶⁻¹⁸G, I, U (Tb46, Figure 1),^16,19−22and V (Tb45, Figure

1)^16,20−24and pretubulysin D (PTb-D43, Figure 1),^8a,11as well

as numerous analogues, have been accomplished.²⁵⁻⁴⁹From

the latter, N¹⁴-desacetoxytubulysin H (Tb1, Figure 1) is

distinguished for its methyl, instead of the acyl methyl,

substituent on N14 of tubulysins A−I^{11,15−18,25}and its high

potency.^6,25We have recently published a total synthesis of

N¹⁴-desacetoxytubulysin H (Tb1, Figure 1) and several of its

analogues (e.g., Tb32, Figure 1) and their biological

delivery systems.⁵²

2. RESULTS AND DISCUSSION

Having developed our C−H activation-based strategy for the

synthesis of the tubuvaline residue,¹¹and in order to devise a

practical synthesis of tubulysins V (Tb45) and U (Tb46),

pretubulysin D (PTb-D43), and their analogues, we decided to

Received: December 1, 2017

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treatment with Me₃SnOH^11,57(cleavage of both methyl ester

and acetate moieties, 68% yield), and re-acetylation of the

resulting hydroxy carboxylic acid (Ac₂O, pyridine, 79% yield) as

shown in Scheme 1.

2.2. Improved Total Synthesis of Pretubulysin D (PTb-

D43) and Its Methyl Ester (PTb-D42). In an eﬀort to

streamline our original synthesis of pretubulysin D (PTb-D43),

and since the adjacent to the thiazole carbonyl moiety was not

needed in this case [cf. intermediates 3 (Scheme 1) and 14

(Scheme 2)], we decided to employ the commercially available

valine derivative 11 as the starting material. Thus, and as shown

in Scheme 2, exposure of 11 to TMSCHN₂followed by LiAlH₄

reduction of the resulting methyl ester furnished the

corresponding primary alcohol, whose reaction with CBr₄and

PPh₃led to bromide 12 in 62% overall yield for the three steps.

Coupling of the anion generated from thiazole 13 (see

Supporting Information for preparation), through the action

of n-BuLi, with bromide 12 furnished 14 in 78% yield.⁵⁸

Transformation of TBS-ether 14 to the desired carboxylic acid

(15) was achieved through desilylation (TBAF) followed by

two-step oxidation of the resulting alcohol (DMP; then

NaClO₂), in 78% overall yield. Coupling of carboxylic acid

15 with aminoester 6¹¹in the presence of HATU and Et₃N led

to amide 16 (82% yield). Removal of the Boc protecting group

from the latter (TFA) followed by coupling of the resulting

amine with acid ﬂuoride 17¹¹furnished peptide 18 (i-Pr₂NEt,

95% overall yield for the two steps) as shown in Scheme 2.

Cleavage of the Fmoc-group from 18 under basic conditions

[N(CH₂CH₂NH₂)₃] and coupling of the so formed amine with

N-methyl-D-pipecolic acid (10)¹¹provided pretubulysin D

precursor PTb-D42 in 72% overall yield. Conversion of PTb-

D42 to pretubulysin D (PTb-D43) was accomplished using the

previously reported conditions (LiOH, 91% yield)¹¹as

presented in Scheme 2.

2.3. Synthesis of Pretubulysin D Analogues PTb-D47,

PTb-D48, and PTb-D49. Scheme 3 summarizes the synthesis

of pretubulysin analogues PTb-D47, PTb-D48, and PTb-D49

from known intermediates 18¹¹and 16,¹¹respectively. Thus,

removal of the Fmoc protecting group from 18

[N(CH₂CH₂NH₂)₃] and coupling of the resulting amine with

carboxylic acid 19 (for its synthesis see Supporting

Information) provided pretubulysin analogue PTb-D47 in

82% overall yield, as shown in Scheme 3A. Removal of the Boc

group from 16 (TFA) followed by coupling of the resulting

amine with acid ﬂuoride 20¹¹gave peptide 21 (i-Pr₂NEt, 95%

overall yield) as shown in Scheme 3B. Cleavage of the Fmoc

group from 21 through the action of N(CH₂CH₂NH₂)₃and

coupling of the resulting amine with either N-methyl-^D-

pipecolic acid (10)¹¹or n-butyl-substituted pipecolic acid 19

provided pretubulysin D analogues PTb-D48 and PTb-D49 in

81% and 76% overall yields, respectively.

Figure 1. Molecular structures of naturally occurring tubulysins V

(Tb45) and U (Tb46), pretubulysin D (PTb-D43), N¹⁴-desacetoxy-

tubulysin H (Tb1), and previously synthesized potent tubulysin

analogue (Tb32).

improve and apply our synthetic technologies to that end. In

pursuing new tubulysin analogues, we applied rational ligand

design based on our previously developed preliminary

structure−activity relationships (SARs)¹¹and the recently

reported X-ray crystallographic analysis regarding the binding

requirements of tubulysin-like molecules to microtubules, their

biological target.⁵³

2.1. Total Synthesis of Tubulysin V (Tb45), Tubulysin

U (Tb46) and Its Methyl Ester (Tb44). Our newly

developed, streamlined total synthesis of the naturally occurring

tubulysins V (Tb45) and U (Tb46), and the methyl ester of

the latter (Tb44), proceeded along an appropriately modiﬁed

and improved synthetic route as shown in Scheme 1.¹¹Thus,

aldehyde 1^11,54was subjected to C−H activation coupling with

thiazole derivative 2 [PhI(OCOCF₃)₂, TMSN₃],^11,55furnishing

coupling product 3 in 56% yield. Stereoselective reduction of

the thiazolyl ketone moiety within 3 with (S)-CBS catalyst in

the presence of BH₃·Me₂S^11,56produced alcohol 4 in 83% yield

and as a single diastereoisomer after chromatographic

puriﬁcation. Elaboration of intermediate 4 to acetoxy carboxylic

acid 5 was achieved through a sequence involving deacetylation

(K₂CO₃, MeOH), two-step selective oxidation of the so

generated primary alcohol (TEMPO, BAIB; then NaClO₂),

and acetylation of the secondary alcohol (Ac₂O, Et₃N) in 78%

overall yield. Coupling of carboxylic acid 5 with ammonium salt

6¹¹in the presence of HATU and Et₃N led to amide 7 (94%

yield). Removal of the Boc group from the latter through the

action of TFA, followed by coupling of the resulting amine with

carboxylic acid 8,¹¹produced peptide 9 (HATU, Et₃N, 92%

yield) as shown in Scheme 1. Cleavage of the Boc protecting

group from 9 (TFA) and coupling of the resulting amine with

N-methyl-^D-pipecolic acid (10) aﬀorded tubulysin U methyl

ester (Tb44, 85% overall yield). Conversion of Tb44 to

tubulysin U (Tb46) via tubulysin V (Tb45) required sequential

2.4. Synthesis of N¹⁴-Desacetoxytubulysin H Ana-

logues Tb50−Tb120. Given that N¹⁴-methyl-substituted

tubulysins (such as N¹⁴-desacetoxytubulysin H,^11,25Tb1)

have been proven more potent than their N¹⁴-H and N¹⁴-

acetoxymethyltubulysin (such as tubulysin H) counterparts, we

focused considerable eﬀorts on designing and synthesizing a

number of N¹⁴-methyl-substituted tubulysins. Scheme 4

summarizes the synthesis of N¹⁴-methyl-substituted tubulysins

Tb50 and Tb51, in which the pipecolic acid residue of the

molecule is replaced with N-Me-substituted pyrrole and

imidazole structural motifs, respectively. Thus, cleavage of the

Fmoc protecting group from previously synthesized inter-

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Figure 2. continued

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Figure 2. Molecular structures of synthesized naturally occurring tubulysins [pretubulysin D (PTb-D43), tubulysins U (Tb46) and V (Tb45)] and

synthesized designed tubulysin analogues (Tb44, PTb-D42, PTb-D47, PTb-D48, PTb-D49, and Tb50−Tb120).

mediate 22¹¹through the action of N,N-bis(2-aminoethyl)-1,2-

ethanediamine followed by coupling of the so generated amine

with 1-methyl-1H-pyrrole-2-carboxylic acid (23) and 1-methyl-

1H-imidazole-2-carboxylic acid (24) provided N¹⁴-desacetox-

ytubulysin analogues Tb50 and Tb51, in 74% and 76% yields,

respectively, as summarized in Scheme 4.

Tubulysin analogues Tb52−Tb55, in which changes in the

two end structural motifs were made while keeping the proven

to be desirable N¹⁴-Me and the i-Pr moieties on the isoleucine

residue, were synthesized as shown in Scheme 5. Thus,

coupling of carboxylic acid 25¹¹with commercially available

ammonium salt 26 in the presence of HATU furnished

dipeptide 27 (84% yield). Exposure of this protected dipeptide

to TFA resulted in removal of the Boc group to aﬀord the

corresponding amine, whose coupling with acid ﬂuoride 20 in

the presence of i-Pr₂NEt in DMF led to the formation of

tripeptide 28 (92% overall yield). Removal of the Fmoc group

from 28 [N(CH₂CH₂NH₂)₃], followed by coupling of the

resulting amine with N-methyl-^D-pipecolic acid (10) and n-

butyl-substituted pipecolic acid 19 (see Supporting Information

for preparation) under HATU conditions, resulted in the

formation of tubulysin analogues Tb52 (72% yield) and Tb54

(77% yield), respectively, as shown in Scheme 5 (see

Supporting Information for further details). Finally, the

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Scheme 1. Syntheses of Tubulysin U Methyl Ester Tb44 and

Tubulysins V (Tb45) and U (Tb46)

Scheme 2. Total Synthesis of Pretubulysin D (PTb-D43) and

Its Methyl Ester Precursor PTb-D42

a

Reagents and conditions: (a) TMSCHN₂(1.2 equiv; 2.0 M in diethyl

ether), toluene:methanol (2:1, v/v), 0 → 23 °C, 0.5 h, 74%; (b)

LiAlH₄(2.0 equiv; 2.0 M in THF), THF, 0 °C, 1 h, 98%; (c) CBr₄

(2.0 equiv), PPh₃(2.0 equiv), benzene, 0 → 10 °C, 1 h, 81%; (d) 13

(1.2 equiv), n-BuLi (1.2 equiv; 2.5 M in hexanes), THF, −78 → 0 °C,

2.5 h, 78%; (e) TBAF (2.0 equiv; 1.0 M in THF), THF, 0 °C, 1 h,

94%; (f) DMP (1.5 equiv), CH₂Cl₂, 23 °C, 15 min, 90%; (g) NaClO₂

(5.4 equiv), NaH₂PO₄·H₂O (12.5 equiv), 2-methyl-2-butene (7.5

equiv), t-BuOH, THF, H₂O, 23 °C, 1 h; 92%; (h) 6 (1.5 equiv),

HATU (3.0 equiv), Et₃N (6.0 equiv), DMF, 0 → 23 °C, 24 h, 82%; (i)

TFA (45 equiv), CH₂Cl₂, 23 °C, 2 h; (j) 17 (4.1 equiv), i-Pr₂NEt (6.2

equiv), DMF, 0 → 23 °C, 18 h, 95% for the two steps; (k)

N(CH₂CH₂NH₂)₃(16 equiv), CH₂Cl₂, 0 → 23 °C, 2 h; (l) 10 (3.0

equiv), HATU (3.0 equiv), Et₃N (6.0 equiv), DMF, 0 → 23 °C, 24 h,

72% for the two steps; (m) LiOH·H₂O (5.0 equiv), THF, H₂O, 23 °C,

24 h, 91%. TBAF = tetra-n-butylammonium ﬂuoride; DMP = Dess−

Martin periodinane; Fmoc = ﬂuorenylmethyloxycarbonyl.

a

Reagents and conditions: (a) 1 (2.0 equiv), 2 (1.0 equiv), TMSN₃

(1.5 equiv), PIFA (1.5 equiv), benzene, 23 °C, 12 h; then 1 (2.0

equiv), TMSN₃(1.5 equiv), PIFA (1.5 equiv), 23 °C, 12 h, 56%; (b)

(S)-CBS (0.2 equiv; 1.0 M in toluene), BH₃·Me₂S (5.0 equiv; 2.0 M in

THF), 0 → 23 °C, 18 h, 83%; (c) K₂CO₃(4.0 equiv), MeOH, 23 °C,

3 h, 95%; (d) TEMPO (0.1 equiv), BAIB (1.0 equiv), CH₂Cl₂, 23 °C,

16 h, 98%; (e) NaClO₂(5.4 equiv), NaH₂PO₄·H₂O (12.5 equiv), 2-

methyl-2-butene (7.5 equiv), t-BuOH, THF, H₂O, 23 °C, 12 h; (f)

Ac₂O (3.0 equiv), Et₃N (3.0 equiv), CH₂Cl₂, 0 → 23 °C, 15 h, 78% for

the two steps; (g) 6 (2.0 equiv), HATU (3.2 equiv), Et₃N (6.0 equiv),

DMF, 0 → 23 °C, 14 h, 94%; (h) TFA (1.75 equiv), CH₂Cl₂, 0 → 23

°C, 6 h; (i) 8 (2.0 equiv), HATU (4.0 equiv), Et₃N (10 equiv), DMF,

0 → 23 °C, 12 h, 92% for the two steps; (j) TFA (1.75 equiv),

CH₂Cl₂, 0 → 23 °C, 6 h; (k) 10 (2.2 equiv), HATU (3.2 equiv), Et₃N

(6.0 equiv), DMF, 0 → 23 °C, 24 h, 85% for the two steps; (l)

Me₃SnOH (10 equiv), CH₂Cl₂, reﬂux, 12 h, 68%; (m) Ac₂O (7.5

equiv), pyridine, 0 → 23 °C, 12 h, 79%. TMS = trimethylsilyl; PIFA =

phenyliodine(III)bis(triﬂuoroacetate); (S)-CBS = (3aS)-tetrahydro-1-

methyl-3,3-diphenyl-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaborole;

TEMPO = (2,2,6,6-tetramethylpiperidin-1-yl)oxyl; BAIB = bis-

(acetoxy)iodo benzene; Ac = acetyl; py = pyridine; THF =

tetrahydrofuran; HATU = 1-[bis(dimethylamino)methylene]-1H-

1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexaﬂuorophosphate; DMF =

N,N-dimethylformamide; TFA = triﬂuoroacetic acid; Boc = tert-

butyloxycarbonyl.

corresponding methyl esters were converted to their carboxylic

acid counterparts Tb53 and Tb55, respectively, through the

sequential action of Me₃SnOH^11,57(cleavage of methyl ester

and acetate moieties) and Ac₂O/pyridine (re-acetylation of

hydroxy group) in 68% and 74% overall yield, respectively, as

presented in Scheme 5.

The syntheses of tubulysin analogues Tb56 and Tb57, where

the “right end” (Tup) and the “left end” (Mep) amino acid

residues of N¹⁴-desacetoxy tubulysin Tb1¹¹were replaced with

structural motifs represented by fragments 33 or 34¹¹and 19

(for their synthesis see Supporting Information), respectively,

are presented in Scheme 6. Thus, removal of the Boc group

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Scheme 3. Synthesis of Pretubulysin D Analogues PTb-D47,

PTb-D48, and PTb−D49

Scheme 4. Synthesis of N¹⁴-Desacetoxytubulysin Analogues

Tb50 and Tb51

a

Reagents and conditions: (a) N(CH₂CH₂NH₂)₃(16 equiv), CH₂Cl₂,

0 → 23 °C, 2 h; (b) 23 or 24 (3.0 equiv), HATU (3.0 equiv), Et₃N

(6.0 equiv), DMF, 0 → 23 °C, 24 h, 74% for the two steps for Tb50

and 76% for the two steps for Tb51.

The latter was converted to the desired keto acid analogue

Tb59 (81% yield) through the action of DMP, as shown in

Scheme 7.

Tubulysin analogues Tb60, Tb61, Tb62, and Tb63,

containing a valine instead of an isoleucine residue, were

synthesized as summarized in Scheme 8. Speciﬁcally, the

previously reported analogue Tb32¹¹was converted to its

carboxylic acid counterpart Tb60 through exposure to

Me₃SnOH^11,57(cleavage of methyl ester and acetate moieties,

70% yield) followed by re-acetylation of the hydroxy acid to

Tb61 (Ac₂O/pyridine, 61% yield). Tb60 was converted to its

keto acid counterpart Tb62, in 78% yield, by DMP oxidation as

shown in Scheme 8. Methyl ester formation from the latter

using TMSCHN₂furnished Tb63 in 71% yield.

Scheme 9 summarizes the synthesis of tubulysin analogues

Tb64 and Tb65, in which the thiazole moiety was replaced

with a pyridine structural motif (while maintaining all the other

structural features of Tb32, one of our most promising

tubulysin analogues).¹¹Their synthesis was initiated with the

removal of the Boc group from dipeptide 35¹¹(TFA) followed

by coupling of the liberated amine with Fmoc-protected acid

ﬂuoride 20¹¹to provide tripeptide 36 (99% yield for the two

steps) as shown in Scheme 9. Cleavage of the Fmoc group

[N(CH₂CH₂NH₂)₃] from this intermediate followed by

coupling of the so generated amine with N-methyl-^D-pipecolic

acid (10) led to tubulysin analogue Tb64 (75% overall yield).

Finally, analogue Tb64 was converted to its carboxylic acid

counterpart Tb65 through the sequential action of

Me₃SnOH^11,57and Ac₂O/pyridine in 68% overall yield, as

shown in Scheme 9.

a

Reagents and conditions: (a) N(CH₂CH₂NH₂)₃(16 equiv), CH₂Cl₂,

0 → 23 °C, 2 h; (b) 19 (3.0 equiv), HATU (3.0 equiv), Et₃N (6.0

equiv), DMF, 0 → 23 °C, 24 h, 82% for the two steps; (c) TFA (40

equiv), CH₂Cl₂, 23 °C, 2 h; (d) 20 (4.0 equiv), i-Pr₂NEt (6.0 equiv),

DMF, 0 → 23 °C, 18 h, 95% for the two steps; (e) N(CH₂CH₂NH₂)₃

(16 equiv), CH₂Cl₂, 0 → 23 °C, 2 h; (f) 10 or 19 (3.0 equiv), HATU

(3.0 equiv), Et₃N (6.0 equiv), DMF, 0 → 23 °C, 24 h, 81% for the two

steps for PTb-D48 and 76% for the two steps for PTb-D49.

from 29¹¹(TFA) followed by reaction of the resulting amine

with acid ﬂuoride 17 in the presence of i-Pr₂NEt in DMF led to

the formation of dipeptide 30 (75% overall yield). The latter

was further treated with N(CH₂CH₂NH₂)₃to remove the

Fmoc group, and the resulting amine was coupled with

pipecolic acid derivative 19 (HATU, Et₃N) to furnish tripeptide

31 in 82% overall yield. Tripeptide 31 was then converted to its

carboxylic acid counterpart (32) through sequential treatment

with Me₃SnOH and Ac₂O as described above for the

conversion of Tb52 to Tb53, in 78% overall yield. Finally,

coupling of 32 with ammonium salt 33 or 34 under HATU

conditions furnished tubulysin analogues Tb56 (71% yield) and

Tb57 (76% yield), respectively, as shown in Scheme 6 (see

Supporting Information for further details).

Tubulysin analogues Tb66, Tb67, and Tb68, in which the

thiazole moiety carries a methyl group, were synthesized from

the known and readily available aldehyde 37^11,54as summarized

in Scheme 10. Thus, C−H activation-based coupling of

aldehyde 37 with methyl thiazole acetate 38,¹¹under the

previously reported conditions [PhI(OCOCF₃)₂, TMSN₃],^11,55

provided ketone 39 in 75% yield. Reduction of thiazolyl ketone

39 with (S)-CBS catalyst in the presence of BH₃·Me₂S^11,56

produced alcohol 40 in 72% yield as a single diastereoisomer

Tubulysin analogue Tb59 in which the acetoxy group of Tb2

was replaced with a carbonyl group, was synthesized from the

previously reported acetoxy ester analogue Tb2¹¹through

hydroxy tubulysin Tb58 as summarized in Scheme 7. Thus,

exposure of Tb2 to Me₃SnOH furnished Tb58 in 78% yield.

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a

Scheme 5. Synthesis of Tubulysin Analogues Tb52−Tb55

Scheme 6. Synthesis of Tubulysin Analogues Tb56 and

a

Tb57

a

Reagents and conditions: (a) TFA (40 equiv), CH₂Cl₂, 0 → 23 °C, 2

h; (b) 17 (4.0 equiv), i-Pr₂NEt (6.0 equiv), DMF, 0 → 23 °C, 18 h,

75% for the two steps; (c) N(CH₂CH₂NH₂)₃(16 equiv), CH₂Cl₂, 0

→ 23 °C, 2 h; (d) 19 (1.5 equiv), HATU (1.5 equiv), Et₃N (3.0

equiv), DMF, 0 → 23 °C, 24 h, 82% for the two steps; (e) Me₃SnOH

(50 equiv), 1,2-dichloroethane, reﬂux, 12 h; (f) Ac₂O (6.0 equiv),

pyridine, 0 → 23 °C, 12 h, 78% for the two steps; (g) 33 or 34 (1.2

equiv), HATU (1.2 equiv), Et₃N (2.4 equiv), DMF, 0 → 23 °C, 18 h,

71% for the two steps for Tb56 and 76% for the two steps for Tb57.

a

Reagents and conditions: (a) 26 (1.5 equiv), HATU (3.0 equiv),

Et₃N (6.0 equiv), DMF, 23 °C, 18 h, 84%; (b) TFA (40 equiv),

CH₂Cl₂, 0 → 23 °C, 2 h; (c) 20 (4.0 equiv), i-Pr₂NEt (6.0 equiv),

DMF, 0 → 23 °C, 18 h, 92% for the two steps; (d) N(CH₂CH₂NH₂)₃

(16 equiv), CH₂Cl₂, 0 → 23 °C, 2 h; (e) 10 (3.0 equiv) or 19 (3.0

equiv), HATU (3.0 equiv), Et₃N (6.0 equiv), DMF, 0 → 23 °C, 24 h,

72% for the two steps for Tb52, 77% for the two steps for Tb54; (f)

Me₃SnOH (50 equiv), 1,2-dichloroethane, reﬂux, 12 h; (g) Ac₂O (6.0

equiv), pyridine, 0 → 23 °C, 12 h, 68% for the two steps for Tb53,

74% for the two steps for Tb55.

hydroxy acid (62% overall yield), as shown in Scheme 10. Keto

acid tubulysin analogue Tb68 was obtained from Tb66 by

treatment with Me₃SnOH followed by oxidation of the

resulting hydroxy acid with Dess−Martin periodinane in 64%

overall yield (Scheme 10).

after chromatographic puriﬁcation. The required elaboration of

alcohol 40 to acetoxy carboxylic acid 41 was achieved through a

sequence involving deacetylation (K₂CO₃, MeOH), selective

oxidation of the resulting primary alcohol (TEMPO, BAIB;

then NaClO₂), and acetylation (Ac₂O, pyridine) of the

remaining secondary alcohol, in 61% overall yield for the four

steps. Coupling of carboxylic acid 41 and ammonium salt 6¹¹in

the presence of HATU and Et₃N led to amide 42 (88% yield).

The Boc protecting group was cleaved from the latter

compound (TFA), and the resulting amine was coupled with

acid ﬂuoride 20¹¹(i-Pr₂NEt, 91%) to aﬀord peptide 43 as

shown in Scheme 10. Removal of the Fmoc group from 43

[N(CH₂CH₂NH₂)₃] followed by coupling of the so generated

amine with N-methyl-^D-pipecolic acid (10) provided tubulysin

analogue Tb66 (65% overall yield). Tubulysin analogue Tb67

was formed from Tb66 through methyl ester hydrolysis

(Me₃SnOH) and acetylation (Ac₂O, pyridine) of the resulting

Scheme 11 summarizes the synthesis of tubulysin analogues

Tb69 (lacking the N-Me substituent), Tb70, and Tb71, the

latter two containing the N-methyl pyrrolidine structural motif

as a substitution for the piperidine residue. Thus, advanced

intermediate 43 (for preparation see Scheme 10) was converted

to its amino counterpart through the action of

N(CH₂CH₂NH₂)₃, and the latter was coupled with Fmoc-

protected pipecolic acid 44 and N-methyl-^D-proline (45) to

aﬀord tubulysin analogues Tb69 and Tb70 in 62% and 82%

overall yields, respectively. Finally, methyl ester Tb70 was

converted to its carboxylic acid counterpart Tb71 through the

sequential action of Me₃SnOH^11,57and Ac₂O/pyridine in 74%

overall yield, as shown in Scheme 11.

Scheme 12 depicts the synthesis of tubulysin analogues Tb72

and Tb73, both of which feature an isopropyl group on the

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Scheme 7. Synthesis of Tubulysin Analogues Tb58 and

Tb59

Scheme 9. Synthesis of Tubulysin Analogues Tb64 and

a

Tb65

a

Reagents and conditions: (a) Me₃SnOH (50 equiv), 1,2-dichloro-

ethane, reﬂux, 12 h, 78%; (b) DMP (1.5 equiv), CH₂Cl₂, 23 °C, 30

min, 81%.

a

Scheme 8. Synthesis of Tubulysin Analogues Tb60−Tb63

a

Reagents and conditions: (a) TFA (45 equiv), CH₂Cl₂, 23 °C, 2 h;

(b) 20 (4.0 equiv), i-Pr₂NEt (6.0 equiv), DMF, 0 → 23 °C, 18 h, 99%

for the two steps; (c) N(CH₂CH₂NH₂)₃(15 equiv), CH₂Cl₂, 0 → 23

°C, 2 h; (d) 10 (3.0 equiv), HATU (3.0 equiv), Et₃N (6.0 equiv),

DMF, 0 → 23 °C, 24 h, 75% for the two steps; (e) Me₃SnOH (50

equiv), 1,2-dichloroethane, reﬂux, 12 h; (f) Ac₂O (4.0 equiv), pyridine,

0 → 23 °C, 12 h, 68% for the two steps.

acid 51 through a sequence involving acetylation (Ac₂O,

pyridine, 82% yield), desilylation (TBAF, 98% yield), and

oxidation (DMP, 89% yield; then NaClO₂, 2-methyl-2-butene,

98% yield). Carboxylic acid 51 was coupled with ammonium

salt 6 under HATU conditions furnishing Boc-protected

segment 52, whose deprotection (Boc removal, TFA) and

union of the resulting amine with acid ﬂuoride 20 under

standard conditions led to fragment 53 in 84% overall yield.

Deprotection of the latter [N(CH₂CH₂NH₂)₃] followed by

coupling of the resulting amine with N-methyl-^D-pipecolic acid

(10) under HATU conditions furnished coveted tubulysin

analogue Tb72 (81% overall yield), and its carboxylic acid

counterpart Tb73 (72% overall yield) upon sequential ester

cleavage and re-acetylation under the standard conditions

mentioned above and summarized in Scheme 12.

Tubulysins Tb74 and Tb75 carry oxygenated pipecolic acid

residues as well as an isopropyl group on their thiazole moiety,

as shown in their structures (see Scheme 13). They were

synthesized from advanced intermediate 53 (for preparation,

see Scheme 12) as shown in Scheme 13. Thus, Fmoc derivative

53 was deprotected [N(CH₂CH₂NH₂)₃], and the resulting

amine was reacted with hydroxy N-methyl pipecolic acid 54

under HATU conditions to aﬀord Tb74 in 69% overall yield.

Analogue Tb75 was generated from Tb74 by DMP oxidation

in 78% yield as shown in Scheme 13.

Retaining the valine moiety instead of the isoleucine residue

just like their Tb74 and Tb75 siblings but lacking the isopropyl

group on their thiazole ring, tubulysins Tb76 and Tb77 feature

oxygenated N-methyl pipecolic acid structural motifs and an

ethyl, rather than a methyl, ester group at the other end of the

molecule. Their synthesis proceeded from N-Boc-protected

thiazolyl carboxylic acid 25¹¹as summarized in Scheme 14.

a

Reagents and conditions: (a) Me₃SnOH (50 equiv), 1,2-dichloro-

ethane, reﬂux, 12 h, 70%; (b) Ac₂O (4.0 equiv), pyridine, 0 → 23 °C,

12 h, 61%; (c) DMP (1.5 equiv), CH₂Cl₂, 0 → 23 °C, 30 min, 78%;

(d) TMSCHN₂(1.2 equiv; 2.0 M in diethyl ether), toluene:methanol

(2:1, v/v), 0 → 23 °C, 1 h, 71%.

thiazole structural motif. Thus, commercially available

bromothiazole ester derivative 46 was reduced to the

corresponding primary alcohol (LiBH₄) and the latter was

silylated (TBSCl, imidazole, 86% yield for the two steps) to

aﬀord bromothiazole 47. The lithio derivative generated from

bromide 47 and n-BuLi was then reacted with Weinreb amide

48 forming ketone 49, whose asymmetric reduction with (S)-

CBS catalyst and BH₃·Me₂S gave, stereoselectively, hydroxy

compound 50. The latter was elaborated to acetoxy carboxylic

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Scheme 10. Synthesis of Tubulysin Analogues Tb66−Tb68

Scheme 11. Synthesis of Tubulysin Analogues Tb69−Tb71

a

Reagents and conditions: (a) N(CH₂CH₂NH₂)₃(15 equiv), CH₂Cl₂,

0 → 23 °C, 2 h; (b) 44 or 45 (3.0 equiv), HATU (3.0 equiv), Et₃N

(6.0 equiv), DMF, 0 → 23 °C, 24 h, 62% for the two steps for Tb69

and 82% for the two steps for Tb70; (c) Me₃SnOH (50 equiv), 1,2-

dichloroethane, reﬂux, 12 h; (d) Ac₂O (4.0 equiv), pyridine, 0 → 23

°C, 12 h, 74% for the two steps.

leading to tripeptide 57. Analogue Tb76 was smoothly

generated from 57, upon liberation of its amino group

[N(CH₂CH₂NH₂)₃] and union of the resulting amine substrate

with hydroxypipecolic acid 54 under the inﬂuence of HATU

and Et₃N (96% yield for the two steps). Finally, silylation of the

resulting alcohol with TBDMSOTf and 2,6-lutidine furnished

analogue Tb77 in 87% overall yield, as presented in Scheme 14.

The latter analogue was meant to test the eﬀect of increased

lipophilicity of the TBS-bearing pipecolic acid residue.

Tubulysin analogue Tb78, whose novel structural motif is the

pentyl spirocycle moiety instead of the isoleucine residue, was

synthesized as shown in Scheme 15. Thus, removal of the Boc

group from previously reported dipeptide 58¹¹(TFA) and

coupling of the liberated amine with Fmoc-protected acid

ﬂuoride 59 (see Supporting Information for preparation) under

standard conditions provided tripeptide 60 (56% yield for the

two steps). Cleavage of the Fmoc group [N(CH₂CH₂NH₂)₃]

from the latter followed by coupling of the generated amine

with N-methyl-^D-pipecolic acid (10) led to the targeted

tubulysin analogue Tb78 in 69% overall yield.

Scheme 16 summarizes the synthesis of tubulysin analogues

Tb79 and Tb80, both of which feature a hexaﬂuoro isopropyl

unit⁵⁹as opposed to their isoleucine residue. Their synthesis

began with removal of the Boc group from the previously

reported dipeptide 58¹¹and proceeded with coupling of the

liberated amine with Fmoc-protected acid ﬂuoride 61

(prepared from its amino acid counterpart by sequential

exposure to FmocCl and DAST; see Supporting Information

for details) followed by cleavage of the Fmoc group

[N(CH₂CH₂NH₂)₃] to aﬀord amine 62 (27% yield for the

three steps) as shown in Scheme 16 (see Supporting

Information for further details). Coupling of the so generated

a

Reagents and conditions: (a) 37 (2.0 equiv), 38 (1.0 equiv), TMSN₃

(2.0 equiv), PIFA (2.0 equiv), benzene, 23 °C, 16 h; then 37 (2.0

equiv), TMSN₃(2.0 equiv), PIFA (2.0 equiv), 23 °C, 12 h, 75%; (b)

(S)-CBS (0.2 equiv; 1.0 M in toluene), BH₃·Me₂S (1.0 equiv; 2.0 M in

THF), 0 → 23 °C, 18 h, 72%; (c) K₂CO₃(4.0 equiv), MeOH, 23 °C,

3 h, 93%; (d) TEMPO (0.1 equiv), BAIB (1.0 equiv), CH₂Cl₂, 23 °C,

16 h, 91%; (e) NaClO₂(5.4 equiv), NaH₂PO₄·H₂O (12.2 equiv), 2-

methyl-2-butene (7.5 equiv), t-BuOH, THF, H₂O, 23 °C, 12 h; (f)

Ac₂O (3.0 equiv), pyridine (3.0 equiv), CH₂Cl₂, 0 → 23 °C, 15 h, 72%

for the two steps; (g) 6 (1.5 equiv), HATU (3.0 equiv), Et₃N (6.0

equiv), DMF, 0 → 23 °C, 24 h, 88%; (h) TFA (45 equiv), CH₂Cl₂, 0

→ 23 °C, 2 h; (i) 20 (4.0 equiv), i-Pr₂NEt (6.0 equiv), DMF, 0 → 23

°C, 18 h, 91% for the two steps; (j) N(CH₂CH₂NH₂)₃(15 equiv),

CH₂Cl₂, 0 → 23 °C, 2 h; (k) 10 (3.0 equiv), HATU (3.0 equiv), Et₃N

(6.0 equiv), DMF, 0 → 23 °C, 24 h, 65% for the two steps; (l)

Me₃SnOH (50 equiv), 1,2-dichloroethane, reﬂux, 12 h; (m) Ac₂O (4.0

equiv), pyridine, 0 → 23 °C, 12 h, 62% for the two steps; (n) DMP

(1.5 equiv), CH₂Cl₂, 0 → 23 °C, 30 min, 64% for the two steps.

Thus, 25 was coupled to ammonium salt 55 in the presence of

HATU and Et₃N to aﬀord dipeptide 56 (81% yield), whose

exposure to TFA led to the corresponding amine. Coupling of

the latter with acid ﬂuoride 20 was facilitated by i-Pr₂NEt

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Scheme 12. Synthesis of Tubulysin Analogues Tb72 and

Tb73

Scheme 13. Synthesis of Tubulysin Analogues Tb74 and

a

Tb75

a

Reagents and conditions: (a) N(CH₂CH₂NH₂)₃(15 equiv), CH₂Cl₂,

0 → 23 °C, 2 h; (b) 54 (3.0 equiv), HATU (3.0 equiv), Et₃N (6.0

equiv), DMF, 0 → 23 °C, 24 h, 69% for the two steps; (c) DMP (1.5

equiv), CH₂Cl₂, 23 °C, 30 min, 78%.

Scheme 17 summarizes the synthesis of tubulysin analogues

Tb81, Tb82, Tb83, and Tb84, which incorporate a triﬂuoro

ethyl moiety,⁵⁹instead of the isoleucine residue. Their synthesis

began with removal of the Boc group from the previously

reported dipeptide 58¹¹, and coupling of the liberated amine

with Fmoc-protected acid ﬂuoride 63 (prepared from its amino

acid counterpart by sequential exposure to FmocCl, and DAST;

see Supporting Information for details) to provide tripeptide 64

(70% yield for the two steps), as shown in Scheme 17 (see

Supporting Information for further details). Cleavage of the

Fmoc group [N(CH₂CH₂NH₂)₃] from this intermediate

aﬀorded free amine 65 (82% yield), which was coupled with

either N-methyl-^D-pipecolic acid (10) or N-methyl-D-proline

(45) to give tubulysin analogues Tb81 (79% yield) or Tb82

(66% yield), respectively. Finally, exposure of Tb81 and Tb82

to Me₃SnOH^11,57furnished analogues Tb83 (82% yield) and

Tb84 (79% yield), respectively, as shown in Scheme 17.

Scheme 18 summarizes the synthesis of tubulysin analogues

Tb85, Tb86, and Tb87, which incorporate an (R)-hexaﬂuoro

isopropyl moiety⁵⁹instead of the (S)-isoleucine residue found

in many of the other designed analogues. Their synthesis began

with removal of the Boc group (TFA) from the previously

reported dipeptide 58¹¹followed by coupling of the so

obtained amine with Fmoc-protected acid ﬂuoride 66

(prepared from its amino acid counterpart by sequential

exposure to FmocCl and DAST) to provide tripeptide 67, upon

cleavage of the Fmoc group [N(CH₂CH₂NH₂)₃] (38% yield

for the three steps), as shown in Scheme 18 (see Supporting

Information for further details). Coupling of the latter with

either N-methyl-^D-pipecolic acid (10) or N-methyl-D-proline

(45) under HATU conditions led to tubulysin analogues Tb85

(89% yield) and Tb86 (88% yield), respectively. Sequential

treatment of Tb85 with Me₃SnOH^11,57and Ac₂O/pyridine

then gave analogue Tb87 in 89% overall yield as shown in

Scheme 18.

a

Reagents and conditions: (a) LiBH₄(1.53 equiv; 2 M in THF),

MeOH (1.55 equiv), THF, 0 → 23 °C, 12 h; (b) TBSCl (1.23 equiv),

imidazole (1.23 equiv), CH₂Cl₂, 0 → 23 °C, 0.5 h, 86% for the two

steps; (c) n-BuLi (1.44 equiv; 2.5 M in hexanes), 48 (1.0 equiv), THF,

−78 → −50 °C, 3 h, 71%; (d) (S)-CBS (0.1 equiv, 1.0 M in toluene),

BH₃·Me₂S (1.0 equiv; 2.0 M in THF), 0 → 23 °C, 36 h, 74%; (e)

Ac₂O (3.0 equiv), Et₃N (4.0 equiv), 0 → 23 °C, 2 h, 82%; (f) TBAF

(2.0 equiv; 1 M in THF), THF, 0 → 23 °C, 30 min, 98%; (g) DMP

(1.5 equiv), CH₂Cl₂, 23 °C, 1 h, 89%; (h) NaClO₂(5.4 equiv),

NaH₂PO₄·H₂O (12.2 equiv), 2-methyl-2-butene (7.5 equiv), t-BuOH,

THF, H₂O, 23 °C, 1 h, 98%; (i) 6 (1.5 equiv), HATU (3.0 equiv),

Et₃N (6.0 equiv), DMF, 0 → 23 °C, 24 h, 99%; (j) TFA (45 equiv),

CH₂Cl₂, 0 → 23 °C, 2 h; (k) 20 (4.0 equiv), i-Pr₂NEt (6.0 equiv),

DMF, 0 → 23 °C, 18 h, 84% for the two steps; (l) N(CH₂CH₂NH₂)₃

(15 equiv), CH₂Cl₂, 0 → 23 °C, 2 h; (m) 10 (3.0 equiv), HATU (3.0

equiv), Et₃N (6.0 equiv), DMF, 0 → 23 °C, 24 h, 81% for the two

steps; (n) Me₃SnOH (50 equiv), 1,2-dichloroethane, reﬂux, 12 h; (o)

Ac₂O (4.0 equiv), pyridine, 0 → 23 °C, 12 h, 72% for the two steps.

amine 62 with N-methyl-D-pipecolic acid (10) resulted in the

formation of tubulysin analogue Tb79 (87% yield). Finally,

time controlled exposure of Tb79 to Me₃SnOH^11,57(5 h;

cleavage of acetate only) furnished analogue Tb80 in 87% yield,

as shown in Scheme 16.

Tubulysin analogue Tb88, lacking isoleucin’s side chain, was

constructed as shown in Scheme 19. Thus, removal of the Boc

group from dipeptide 56 (for preparation, see Scheme 14) with

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Scheme 14. Synthesis of Tubulysin Analogues Tb76 and

Tb77

Scheme 15. Synthesis of Tubulysin Analogue Tb78

a

Reagents and conditions: (a) TFA (45 equiv), CH₂Cl₂, 0 → 23 °C, 2

h; (b) 59 (4.0 equiv), i-Pr₂NEt (6.0 equiv), DMF, 0 → 23 °C, 18 h,

56% for the two steps; (c) N(CH₂CH₂NH₂)₃(15 equiv), CH₂Cl₂, 0

→ 23 °C, 2 h; (d) 10 (3.0 equiv), HATU (3.0 equiv), Et₃N (6.0

equiv), DMF, 0 → 23 °C, 24 h, 69% for the two steps.

Scheme 16. Synthesis of Tubulysin Analogues Tb79 and

a

Tb80

a

Reagents and conditions: (a) 55 (1.5 equiv), HATU (3.0 equiv),

Et₃N (6.0 equiv), DMF, 23 °C, 24 h, 81%; (b) TFA (45 equiv),

CH₂Cl₂, 0 → 23 °C, 2 h; (c) 20 (4.0 equiv), i-Pr₂NEt (6.0 equiv),

DMF, 0 → 23 °C, 18 h, 99% for the two steps; (d) N(CH₂CH₂NH₂)₃

(15 equiv), CH₂Cl₂, 0 → 23 °C, 2 h; (e) 54 (3.0 equiv), HATU (3.0

equiv), Et₃N (6.0 equiv), DMF, 0 → 23 °C, 24 h, 96% for the two

steps; (f) TBDMSOTf (2.0 equiv), 2,6-lutidine (3.0 equiv), CH₂Cl₂, 0

→ 23 °C, 0.5 h, 87%.

TFA followed by coupling of the resulting amine with Fmoc-

protected acid ﬂuoride 68 in the presence of i-Pr₂NEt aﬀorded

tripeptide 69 (86% yield for the two steps). Cleavage of the

Fmoc group [N(CH₂CH₂NH₂)₃] from the latter followed by

coupling of the generated amine with N-methyl-^D-pipecolic acid

(10) under HATU conditions led to the targeted tubulysin

analogue Tb88 (72% overall yield).

Scheme 20 summarizes the synthesis of tubulysin analogues

Tb89 and Tb91, both featuring an alanine in place of their

isoleucine residue, and Tb90 and Tb92, which furthermore

feature the proline counterpart (as represented by building

block 45) of the pipecolic acid residue. The synthesis of these

tubulysin analogues started with Boc-protected dipeptide 58¹¹

and proceeded through tripeptide 71. Thus, exposure of 58 to

TFA generated the corresponding free amine, which was

coupled with acid ﬂuoride 70 (prepared from its amino acid

precursor by sequential treatment with FmocCl and DAST; see

Supporting Information for further details) in the presence of i-

Pr₂NEt to furnish 71 in 92% overall yield. Removal of the Fmoc

group [N(CH₂CH₂NH₂)₃] from this intermediate followed by

union of the resulting amine with either N-methyl-^D-pipecolic

acid (10) or N-methyl-^D-proline (45) in the presence of HATU

led to tubulysin analogues Tb89 (89% overall yield) and Tb90

a

Reagents and conditions: (a) TFA (45 equiv), CH₂Cl₂, 0 → 23 °C, 2

h; (b) 61 (4.0 equiv), i-Pr₂NEt (6.0 equiv), DMF, 0 → 23 °C, 18 h;

(c) N(CH₂CH₂NH₂)₃(15 equiv), CH₂Cl₂, 0 → 23 °C, 2 h, 27% for

the three steps; (d) 10 (3.0 equiv), HATU (3.0 equiv), Et₃N (6.0

equiv), DMF, 0 → 23 °C, 24 h, 87%; (e) Me₃SnOH (20 equiv), 1,2-

dichloroethane, reﬂux, 5 h, 87%.

(88% overall yield), respectively. Finally, Tb89 and Tb90 were

converted to their carboxylic acid counterparts Tb91 and Tb92

through the sequential action of Me₃SnOH^11,57and Ac₂O/

pyridine in 82% and 85% overall yield, respectively, as

presented in Scheme 20.

Tubulysin analogues Tb93 and Tb94, featuring an ethyl

group instead of the isobutyl group at their isoleucine residue,

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Scheme 17. Synthesis of Tubulysin Analogues Tb81−Tb84

Scheme 18. Synthesis of Tubulysin Analogues Tb85−Tb87

a

Reagents and conditions: (a) TFA (45 equiv), CH₂Cl₂, 0 → 23 °C, 2

h; (b) 66 (4.0 equiv), i-Pr₂NEt (6.0 equiv), DMF, 0 → 23 °C, 18 h;

(c) N(CH₂CH₂NH₂)₃(15 equiv), CH₂Cl₂, 0 → 23 °C, 2 h, 38% for

the three steps; (d) 10 or 45 (3.0 equiv), HATU (3.0 equiv), Et₃N

(6.0 equiv), DMF, 0 → 23 °C, 24 h, 89% for Tb85 and 88% for Tb86;

(e) Me₃SnOH (50 equiv), 1,2-dichloroethane, reﬂux, 12 h; (f) Ac₂O

(4.0 equiv), pyridine, 0 → 23 °C, 12 h, 89% for the two steps.

a

Scheme 19. Synthesis of Tubulysin Analogue Tb88

a

Reagents and conditions: (a) TFA (45 equiv), CH₂Cl₂, 0 → 23 °C, 2

h; (b) 63 (4.0 equiv), i-Pr₂NEt (6.0 equiv), DMF, 0 → 23 °C, 18 h,

70% for the two steps; (c) N(CH₂CH₂NH₂)₃(15 equiv), CH₂Cl₂, 0

→ 23 °C, 2 h, 82%; (d) 10 or 45 (3.0 equiv), HATU (3.0 equiv), Et₃N

(6.0 equiv), DMF, 0 → 23 °C, 24 h, 79% for Tb81 and 66% for Tb82;

(e) Me₃SnOH (20 equiv), 1,2-dichloroethane, reﬂux, 5 h, 82% for the

Tb83 and 79% for the Tb84.

were synthesized as summarized in Scheme 21. Thus, removal

of the Boc group from fragment 56 (for preparation, see

Scheme 14) with TFA and coupling of the so obtained amine

with Fmoc-protected acid ﬂuoride 72 in the presence of i-

Pr₂NEt provided tripeptide 73 (85% yield for the two steps).

Cleavage of the Fmoc group [N(CH₂CH₂NH₂)₃] from the

latter followed by coupling of the resulting amine with N-

methyl-^D-pipecolic acid (10) gave tubulysin analogue Tb93

(86% overall yield). Methyl ester Tb93 was then converted to

its carboxylic acid counterpart Tb94 through sequential use of

Me₃SnOH^11,57and Ac₂O/pyridine in 76% overall yield, as

shown in Scheme 21.

Scheme 22 summarizes the synthesis of tubulysin analogues

Tb95 and Tb96, whose primary feature is the n-butyl group in

place of their isoleucine side chain. Their synthesis began with

removal of the Boc group from intermediate 56 (for

preparation, see Scheme 14) and coupling of the liberated

amine with Fmoc-protected acid ﬂuoride 74 (prepared from its

a

Reagents and conditions: (a) TFA (45 equiv), CH₂Cl₂, 0 → 23 °C, 2

h; (b) 68 (4.0 equiv), i-Pr₂NEt (6.0 equiv), DMF, 0 → 23 °C, 18 h,

86% for the two steps; (c) N(CH₂CH₂NH₂)₃(15 equiv), CH₂Cl₂, 0

→ 23 °C, 2 h; (d) 10 (3.0 equiv), HATU (3.0 equiv), Et₃N (6.0

equiv), DMF, 0 → 23 °C, 24 h, 72% for the two steps.

amino acid counterpart by sequential exposure to FmocCl and

DAST) to provide tripeptide 75 (98% yield for the two steps)

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Scheme 20. Synthesis of Tubulysin Analogues Tb89−Tb92

Scheme 21. Synthesis of Tubulysin Analogues Tb93 and

a

Tb94

a

Reagents and conditions: (a) TFA (45 equiv), CH₂Cl₂, 0 → 23 °C, 2

h; (b) 72 (4.0 equiv), i-Pr₂NEt (6.0 equiv), DMF, 0 → 23 °C, 18 h,

85% for the two steps; (c) N(CH₂CH₂NH₂)₃(15 equiv), CH₂Cl₂, 0

→ 23 °C, 2 h; (d) 10 (3.0 equiv), HATU (3.0 equiv), Et₃N (6.0

equiv), DMF, 0 → 23 °C, 24 h, 86% for the two steps; (e) Me₃SnOH

(50 equiv), 1,2-dichloroethane, reﬂux, 12 h; (f) Ac₂O (4.0 equiv),

pyridine, 0 → 23 °C, 12 h, 76% for the two steps.

Scheme 22. Synthesis of Tubulysin Analogues Tb95 and

a

Tb96

a

Reagents and conditions: (a) TFA (45 equiv), CH₂Cl₂, 0 → 23 °C, 2

h; (b) 70 (4.0 equiv), i-Pr₂NEt (6.0 equiv), DMF, 0 → 23 °C, 18 h,

92% for the two steps; (c) N(CH₂CH₂NH₂)₃(15 equiv), CH₂Cl₂, 0

→ 23 °C, 2 h; (d) 10 or 45 (3.0 equiv), HATU (3.0 equiv), Et₃N (6.0

equiv), DMF, 0 → 23 °C, 24 h, 89% for the two steps for Tb89 and

88% for the two steps for Tb90; (e) Me₃SnOH (50 equiv), 1,2-

dichloroethane, reﬂux, 12 h; (f) Ac₂O (4.0 equiv), pyridine, 0 → 23

°C, 12 h, 82% for the two steps for Tb91 and 85% for the two steps

for Tb92.

as shown in Scheme 22 (see Supporting Information for further

details). Cleavage of the Fmoc group [N(CH₂CH₂NH₂)₃] from

this intermediate, followed by coupling with either N-methyl-^D-

pipecolic acid (10) or its proline sibling 45 led to tubulysin

analogues Tb95 (90% overall yield) and Tb96 (87% overall

yield), respectively, as shown in Scheme 22.

Tubulysin analogues Tb97 and Tb98, whose novel feature is

their 3-methylbutyl moiety as opposed to their isoleucine

residue, were constructed from dipeptide fragment 56 (for

preparation see Scheme 14). Thus, removal of the Boc group

from dipeptide 56 and coupling of the so generated amine with

Fmoc-protected acid ﬂuoride 76 (prepared from its amino acid

counterpart by sequential exposure to FmocCl and DAST)

provided tripeptide 77 (93% yield for the two steps) as shown

in Scheme 23 (see Supporting Information for further details).

Cleavage of the Fmoc group [N(CH₂CH₂NH₂)₃] from the

latter followed by coupling with either N-methyl-^D-pipecolic

acid (10) or N-methyl-^D-proline (45) led to tubulysin

analogues Tb97 (79% overall yield) or Tb98 (74% overall

yield), as summarized in Scheme 23.

a

Reagents and conditions: (a) TFA (45 equiv), CH₂Cl₂, 0 → 23 °C, 2

h; (b) 74 (4.0 equiv), i-Pr₂NEt (6.0 equiv), DMF, 0 → 23 °C, 18 h,

98% for the two steps; (c) N(CH₂CH₂NH₂)₃(15 equiv), CH₂Cl₂, 0

→ 23 °C, 2 h; (d) 10 or 45 (3.0 equiv), HATU (3.0 equiv), Et₃N (6.0

equiv), DMF, 0 → 23 °C, 24 h, 90% for the two steps for Tb95 and

87% for the two steps for Tb96.

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Scheme 23. Synthesis of Tubulysin Analogues Tb97 and

Tb98

Scheme 24. Synthesis of Tubulysin Analogues Tb99−

a

Tb101

a

Reagents and conditions: (a) 79 or 80 or 81 (5.0 equiv), HATU (5.0

equiv), Et₃N (10 equiv), DMF, 0 → 23 °C, 16 h, 70% for Tb99, 72%

for Tb100, and 75% for Tb101.

a

Reagents and conditions: (a) TFA (45 equiv), CH₂Cl₂, 0 → 23 °C, 2

h; (b) 76 (4.0 equiv), i-Pr₂NEt (6.0 equiv), DMF, 0 → 23 °C, 18 h,

93% for the two steps; (c) N(CH₂CH₂NH₂)₃(15 equiv), CH₂Cl₂, 0

→ 23 °C, 2 h; (d) 10 or 45 (3.0 equiv), HATU (3.0 equiv), Et₃N (6.0

equiv), DMF, 0 → 23 °C, 24 h, 79% for the two steps for Tb97 and

74% for the two steps for Tb98.

fragment 56 (prepared as shown in Scheme 14) as summarized

in Scheme 26. Thus, deprotection of the amino group (TFA) of

56 and coupling of the resulting amine with acid ﬂuoride 86

(see Supporting Information for preparation) in the presence of

i-Pr₂NEt aﬀorded tripeptide 87 (81% overall yield for the two

steps). Removal of the Fmoc group from the latter

[N(CH₂CH₂NH₂)₃] and coupling of the so generated amine

with carboxylic acid 10¹¹(HATU, Et₃N) led ﬁrst to Tb106

(76% overall yield) and then to Tb107 upon ester hydrolysis

(Me₃SnOH)^11,57and re-acetylation (Ac₂O, pyridine), in 84%

yield for the two steps.

Tubulysin analogues Tb108 and Tb109, carrying a 3,3-

dimethylpentanoic group at their isoleucine residue were

similarly synthesized from 56 as depicted in Scheme 27.

Thus, deprotection of 56 as described above (i.e., TFA)

followed by i-Pr₂NEt facilitated coupling of the resulting amine

with acid ﬂuoride 88 (see Supporting Information for

preparation) furnished intermediate tripeptide 89 (72% overall

yield from 56). Fmoc removal from 89 with [N-

(CH₂CH₂NH₂)₃] and coupling of the so obtained amine

with carboxylic acid 10 facilitated by HATU and Et₃N led, in

74% overall yield, to analogue Tb108. Finally, exposure of

Tb108 to Me₃SnOH furnished the corresponding hydroxy

carboxylic acid, which was acetylated (Ac₂O, pyridine) to aﬀord

analogue Tb109, in 70% overall yield from Tb108, as shown in

Scheme 27.

Tubulysins Tb99−Tb101 are characterized with rather

drastic modiﬁcations at their isoleucine and tubuphenylalanine

residues (i.e., cyclopropyl, cubane, and [1.1.1]bicyclopentane

moieties). Their syntheses are shown in Scheme 24. Thus, the

previously synthesized cyclopropyl-carrying intermediate 78¹¹

was coupled with amino esters 79,^11,6080,^11,60and 81¹¹under

the inﬂuence of HATU and Et₃N to aﬀord amides Tb99 (70%

yield), Tb100 (72% yield) and Tb101 (75% yield),

respectively.

Tubulysin analogues Tb102 and Tb103 (both featuring a

[1.1.1]bicyclopentane structural motif at the “right edge” of the

molecule instead of the tubuphenylalanine residue), Tb104

(featuring the bulkier naphthalene instead of the phenyl moiety

on its tubuphenylalanine residue), and Tb105 (missing the

methyl group on its tubuphenylalanine residue) were

synthesized as highlighted in Scheme 25. Thus, key

intermediate 82¹¹was coupled with amino acid methyl ester

83 (see Supporting Information for its preparation) under the

inﬂuence of HATU and Et₃N to aﬀord Tb103 (79% yield).

Similarly carboxylic acid 82¹¹was joined with amino acid

methyl ester 80^11,60leading to Tb102 (75% yield). Tb104 and

Tb105 were synthesized through similar couplings of 82¹¹with

fragments 84 (see Supporting Information for preparation)

(78% yield) and 85 (see Supporting Information for

preparation) (78% yield) as summarized in Scheme 25.

The next series of tubulysin analogues (i.e., Tb106−Tb109,

Schemes 26 and 27) were intended to probe the eﬀect of shape,

but mainly volume of the lipophilic substituent of the isoleucine

residue on the potency of the tubulysin molecule. Thus,

tubulysins Tb106 and Tb107, carrying a tertiary butyl group on

their isoleucine residue, were synthesized from dipeptide

In an attempt to decipher further SARs within the tubulysin

family of compounds we designed and synthesized tubulysin

analogues Tb110−Tb113 (Scheme 28) equipped with

benzyloxy ethyl and hydroxy ethyl groups on the thiazole

ring. To this end, bromothiazole methyl ester 90⁶¹(for

preparation, see Supporting Information) was reduced with

DIBAL-H to the corresponding alcohol, which was silylated

(TBSOTf, 2,6-lutidine) to aﬀord TBS-ether 91 (86% overall

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Scheme 25. Synthesis of Tubulysin Analogues Tb102−

Scheme 26. Synthesis of Tubulysin Analogues Tb106 and

Tb107

a

Tb105

a

Reagents and conditions: (a) TFA (45 equiv), CH₂Cl₂, 0 → 23 °C, 2

h; (b) 86 (4.0 equiv), i-Pr₂NEt (6.0 equiv), DMF, 0 → 23 °C, 18 h,

81% for the two steps; (c) N(CH₂CH₂NH₂)₃(15 equiv), CH₂Cl₂, 0

→ 23 °C, 2 h; (d) 10 (3.0 equiv), HATU (3.0 equiv), Et₃N (6.0

equiv), DMF, 0 → 23 °C, 24 h, 76% for the two steps; (e) Me₃SnOH

(50 equiv), 1,2-dichloroethane, reﬂux, 12 h; (f) Ac₂O (4.0 equiv),

pyridine, 0 → 23 °C, 12 h, 84% for the two steps.

Scheme 27. Synthesis of Tubulysin Analogues Tb108 and

Tb109

a

Reagents and conditions: (a) 80 or 83 or 84 or 85 (1.2 equiv),

HATU (1.2 equiv), Et₃N (2.4 equiv), DMF, 0 → 23 °C, 18 h, 75% for

Tb102, 79% for Tb103, 78% for Tb104, and 78% for Tb105.

yield). Bromide 91 was treated with n-BuLi, and to the resulting

lithio derivative was added Weinreb amide 92¹¹to aﬀord

ketone 93 in 70% yield. Reduction of the latter with BH₃·Me₂S

in the presence of CBS catalyst^11,56furnished stereoselectively

hydroxy compound 94 (66% yield). The latter compound was

elaborated to acetoxy carboxylic acid 95 through a sequence

involving acetylation (Ac₂O, Et₃N, 88% yield), desilylation

(TBAF, 99% yield), and oxidation (DMP, 91% yield; then

NaClO₂, 99% yield). Coupling carboxylic acid 95 with

ammonium salt 55 through the action of HATU and Et₃N

furnished fragment 96 in 93% yield. Removal of the Boc group

(TFA) from the latter followed by coupling of the so generated

amine with acid ﬂuoride 20 gave tripeptide 97 (92% overall

yield). Finally, cleavage of the Fmoc group from 97 and

coupling of the resulting amine with carboxylic acid 10

facilitated by HATU and Et₃N led to analogue Tb110 in

75% overall yield. Tubulysin analogue Tb111 was obtained

from Tb110 through the standard procedure of hydrolysis

(acetate and ethyl ester) with LiOH¹¹followed by re-

acetylation of the resulting hydroxy acid with Ac₂O/pyridine

(77% overall yield). Finally, Tb110 and Tb111 were converted

to Tb112 and Tb113 in 71% and 65% yield, respectively,

through hydrogenolysis [Pd(OH)₂/C catalyst, H₂] as shown in

Scheme 28.

a

Reagents and conditions: (a) TFA (45 equiv), CH₂Cl₂, 0 → 23 °C, 2

h; (b) 88 (4.0 equiv), i-Pr₂NEt (6.0 equiv), DMF, 0 → 23 °C, 18 h,

72% for the two steps; (c) N(CH₂CH₂NH₂)₃(15 equiv), CH₂Cl₂, 0

→ 23 °C, 2 h; (d) 10 (3.0 equiv), HATU (3.0 equiv), Et₃N (6.0

equiv), DMF, 0 → 23 °C, 24 h, 74% for the two steps; (e) Me₃SnOH

(50 equiv), 1,2-dichloroethane, reﬂux, 12 h; (f) Ac₂O (4.0 equiv),

pyridine, 0 → 23 °C, 12 h, 70% for the two steps.

We ﬁnally explored modiﬁcations at the tubuphenylalanine

(Tup) site of the tubulysin molecule as demonstrated with the

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Scheme 28. Synthesis of Tubulysin Analogues Tb110−

Scheme 29. Synthesis of Tubulysin Analogue Tb114

a

Tb113

a

Reagents and conditions: (a) 98 (1.2 equiv), HATU (1.2 equiv),

Et₃N (2.4 equiv), DMF, 0 → 23 °C, 18 h, 54%.

Scheme 30. Synthesis of Tubulysin Analogues Tb115−

a

Tb118

a

Reagents and conditions: (a) DIBAL-H (3.0 equiv; 1.0 M in diethyl

ether), diethyl ether, −78 → 23 °C, 1 h, 87%; (b) TBSOTf (1.2

equiv), 2,6-lutidine (2.0 equiv), CH₂Cl₂, 0 → 23 °C, 0.5 h, 99%; (c) n-

BuLi (1.44 equiv; 2.5 M in hexanes), 92 (1.0 equiv), THF, −78 →

−50 °C, 3 h, 70%; (d) (S)-CBS (0.1 equiv; 1.0 M in toluene), BH₃·

Me₂S (1.0 equiv; 2.0 M in THF), 0 → 23 °C, 36 h, 66%; (e) Ac₂O

(3.0 equiv), Et₃N (4.0 equiv), 0 → 23 °C, 2 h, 88%; (f) TBAF (2.0

equiv; 1.0 M in THF), THF, 0 → 23 °C, 30 min, 99%; (g) DMP (1.5

equiv), CH₂Cl₂, 23 °C, 0.5 h, 91%; (h) NaClO₂(5.4 equiv),

NaH₂PO₄·H₂O (12.2 equiv), 2-methyl-2-butene (7.5 equiv), t-BuOH,

THF, H₂O, 23 °C, 1 h, 99%; (i) 55 (1.5 equiv), HATU (3.0 equiv),

Et₃N (6.0 equiv), DMF, 0 → 23 °C, 24 h, 93%; (j) TFA (45 equiv),

CH₂Cl₂, 0 → 23 °C, 2 h; (k) 20 (4.0 equiv), i-Pr₂NEt (6.0 equiv),

DMF, 0 → 23 °C, 18 h, 92% for the two steps; (l) N(CH₂CH₂NH₂)₃

(15 equiv), CH₂Cl₂, 0 → 23 °C, 2 h; (m) 10 (3.0 equiv), HATU (3.0

equiv), Et₃N (6.0 equiv), DMF, 0 → 23 °C, 24 h, 75% for the two

steps; (n) LiOH·H₂O (5.0 equiv), THF:H₂O (5:1, v/v), 23 °C, 24 h;

(o) Ac₂O (4.0 equiv), pyridine, 0 → 23 °C, 12 h, 77% for the two

steps; (p) Pd(OH)₂/C (20 wt%), H₂, MeOH, 23 °C, 18 h, 71% for

Tb112 and 65% for Tb113. DIBAL-H = diisobutylaluminum hydride.

a

Reagents and conditions: (a) 99 (ca. 4:1 dr, 1.2 equiv), HATU (1.2

equiv), Et₃N (2.4 equiv), DMF, 0 → 23 °C, 18 h, 26% for Tb115 and

52% for Tb116; (b) 10% Pd/C (50 wt%), H₂, MeOH, 23 °C, 20 h,

88%; (c) 10% Pd/C (50 wt%), H₂, wet EtOH, 23 °C, 20 h, 77%.

structures of analogues Tb114−Tb118 (Schemes 29 and 30).

Tubulysin analogue Tb114, possessing a C₂-symmetric malonic

acid type structural motif at its “right end” (resembling

Meldrum’s acid structure) was synthesized from carboxylic

acid 82¹¹and ammonium salt 98⁶²(see Supporting

Information for details) through the action of HATU in the

presence of Et₃N in 54% yield, as shown in Scheme 29.

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The amino containing tubulysin analogues Tb115−Tb118

were prepared from the previously synthesized fragment 82¹¹

and amino acid derivative 99⁶³(diastereomeric mixture ca. 4:1,

see Supporting Information for preparation) as summarized in

Scheme 30. Thus, coupling of carboxylic acid 82 with

ammonium salt 99 in the presence of HATU and Et₃N yielded

Tb115 and Tb116 as a mixture of diastereoisomers (78% yield,

ca. 1:2, separated by silica gel column chromatography). Tb116

was subjected to hydrogenolysis in MeOH (10% Pd/C, 50

wt%, H₂, 23 °C, 20 h) to aﬀord dimethylamino tubulysin

analogue Tb117 in 88% yield. Similar treatment of Tb116 in

EtOH led to ethyl amino tubulysin analogue Tb118 in 77%

yield. Apparently, this is a known outcome of hydrogenolytic

Cbz group cleavage from primary amines under strong catalyst

and prolonged time conditions.⁶⁴As demonstrated here, this

reaction provides practical means for accessing substituted

amines of considerable complexity.

Scheme 32. Synthesis of Tubulysin Analogues Tb119

Scheme 31 summarizes the synthesis of Tb119 and Tb120,

both containing a hydroxymethyl group adjacent to their

Scheme 31. Synthesis of Tubulysin Analogues Tb119 and

a

Tb120

a

Reagents and conditions: (a) TFA (45 equiv), CH₂Cl₂, 0 °C, 3 h,

quant; (b) TfN₃(3.0 equiv; 0.57 M in CH₂Cl₂), CuSO₄·5H₂O (0.1

equiv), K₂CO₃(2.0 equiv), MeOH, H₂O, 23 °C, 12 h, 81% yield for

the two steps; (c) Et₃N (1.8 equiv), LiCl (1.7 equiv), PivCl (1.5

equiv), (S)-4-benzyl-2-oxazolidinone 102 (1.7 equiv), −20 °C, 2 h,

56%; (d) TiCl₄(1.1 equiv), i-Pr₂NEt (1.1 equiv), 1,3,5-trioxane (1.1

equiv), 0 °C, 3.5 h, 54% for 104 and 28% for 105; (e) NaOMe (1.0

equiv), MeOH, CH₂Cl₂, −78 → 0 °C, 2 h, 95%; (f) 10% Pd/C (50 wt

%), H₂, HCl (1.2 equiv; 1 M in MeOH), MeOH, 23 °C, 0.5 h, 99%;

(g) 82 (0.9 equiv), HATU (1.2 equiv), Et₃N (2.4 equiv), DMF, 0 →

23 °C, 18 h, 95%; (h) NaOMe (1.0 equiv), MeOH, CH₂Cl₂, −78 → 0

°C, 2 h, 78%.

a

Reagents and conditions: (a) 33 (1.2 equiv), HATU (1.2 equiv),

Et₃N (2.4 equiv), DMF, 0 → 23 °C, 18 h, 60% for Tb119 and 18% for

Tb120.

104 (54% yield) and, unexpectedly, its C2 epimeric sibling 105

(28% yield). Besides NMR spectroscopic analyses revealing its

general structure, the absolute stereochemistry of the latter

compound was established by converting it to hydroxy ester

108 (NaOMe; 78% yield), whose data matched those obtained

from another sample of the same compound prepared from

oxazolidinone 111 as shown in Scheme 33. Hydroxy azide

methyl ester 106 was then generated from oxazolidinone 104

through the action of NaOMe (95% yield). Reduction (H₂/Pd

catalyst, HCl, MeOH, 99% yield) of the azide group within the

latter provided hydroxy ammonium salt 107, which was

smoothly coupled with tripeptide carboxylic acid 82¹¹under

HATU conditions to aﬀord the targeted tubulysin analogue

Tb119 in 95% yield, as summarized in Scheme 32. The

stereoselective synthesis of diastereomeric analogue Tb120

proceeded through the same sequence, starting with carboxylic

acid 101 and via intermediates 110, 111, 108, and 112, by

utilizing the enantiomeric chiral auxiliary [109: (R)-4-benzyl-2-

oxazolidinone] in similar yields and without the formation of

carboxylate moiety (instead of a methyl group) of the

tubuphenylalanine residue. Thus, tripeptide carboxylic acid

82¹¹was coupled with ammonium salt 33 (ca. 4:1 dr, see

Supporting Information for preparation) under the inﬂuence of

HATU and Et₃N to aﬀord tubulysin analogues Tb119 and

Tb120 as a mixture of diastereoisomers, which were separated

by HPLC to give pure Tb119 (60% yield) and Tb120 (18%

yield).⁶⁴

Stereoselective syntheses of tubulysin analogues Tb119 and

Tb120 were also developed starting with readily available

phenylalanine derivative 100, as shown in Schemes 32 and 33.

Thus, substrate 100 was converted to its azide counterpart 101

through a two-step sequence (TFA; TfN₃, CuSO₄catalyst, 81%

overall yield, Scheme 32). The latter was reacted with chiral

auxiliary 102 [(S)-4-benzyl-2-oxazolidinone] in the presence of

PivCl, Et₃N, and LiCl to aﬀord oxazolidinone 103 (56% yield),

which was hydroxymethylated stereoselectively with trioxane in

the presence of TiCl₄, leading to hydroxy azide oxazolidinone

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binding through a salt bridge to a carboxylate moiety of

tubulin); (2) one amide NH moiety binding through H-

bonding to a carbonyl O of the receptor; (3) a hydrophobic

moiety (i.e., 2-methyl butyl group) binding to a hydrophobic

pocket within tubulin; (4) carbonyl O binding through H-

bonding to an amide NH moiety of tubulin; (5) a second

hydrophobic group (i.e., isopropyl moiety) binding to a

diﬀerent hydrophobic pocket within the tubulin unit; (6) the

thiazole N and the adjacent carbonyl O, both serving as H-

bond acceptors from a H-bond donor on the tubulin unit; (7)

the phenyl moiety of the tubuphenyl alanine residue ﬁtting

snuggly into a hydrophobic cavity within a tubulin unit; and (8)

the carboxylate unit forming a salt bridge with a counterpart

within the tubulin receptor. This model seems to be, more or

less, in accordance with our ﬁndings, correlating well structural

motifs with potencies within the family of compounds

synthesized and tested in this study (see Figure 2 and Table 1).

As the pipecolic acid residue of the tubulysin molecule

perfectly occupies its binding site on tubulin, according to the

X-ray-generated model,⁵³it was not surprising that certain

modiﬁcations made to this fragment led to only insigniﬁcant or

low biological activity. This phenomenon is clearly demon-

strated by tubulysin analogues Tb50 and Tb51 (in which the

pipecolic acid was exchanged for 1-methyl-1H-pyrrole-2-

carboxylic acid). It was also evident in the cases of PTb-D47,

PTb-D49, Tb54, Tb55, and Tb77, in which the pipecolic acid

residue was modiﬁed at the N-atom with the larger n-butyl in

place of the methyl group or oxygenated on one of the carbons

of the ring, changing its steric and/or hydrophobic require-

ments that apparently do not ﬁt the binding site of the tubulin

receptor. In addition, when the pipecolic acid moiety was

replaced with its ﬁve-membered proline counterpart, as in

Tb70 and Tb71, signiﬁcant loss of potency was observed (see

Table 1), providing further support for the strict and crucial

requirements of the pipecolic acid binding site within the

tubulin receptor, although other novel substituents on this

nitrogen-bearing residue may prove fruitful.

As can be seen in Figure 3, the isoleucine (Ile) residue of the

tubuysin ligand provides a hydrogen-bonding opportunity (one

acceptor and one donor) and one hydrophobic moiety (the 3-

methybutyl group) that ﬁts snuggly into the α2 tubulin subunit.

As shown from our results, the tolerance of the hydrophobic

acceptor site is rather limited. Thus, tubulysins equipped with

an isopropyl (e.g., Tb61), tertiary butyl (e.g., Tb106, Tb107),

and the one-carbon higher 1,1-dimethylpropyl moieties (e.g.,

Tb108, Tb109) exhibited exceptional potencies. On the other

hand, the longer n-butyl (e.g., Tb95, Tb96) groups and 3-

methylbutyl (e.g., Tb97, Tb98) instead of the isoleucine side

chain are not tolerated, as evident from the lack of or

signiﬁcantly lower cytotoxicities of the corresponding analogues

(see Table 1). Furthermore, smaller size group substitutions at

the isoleucine side chain position, as in Tb88 (hydrogen) and

Tb89−Tb92 (methyl group), led, surprisingly, to no signiﬁcant

activity. Interestingly, the ethyl group containing tubulysin

analogues Tb93 and Tb94 exhibited signiﬁcant potencies,

indicating perhaps the lower limit of lipophilicity and steric

demand required at that position for potent activity. The

ﬂuorinated tubulysin analogues Tb79−Tb87 carrying sub-

stituted ethyl or isopropyl moieties on the isoleucine residue,

were disappointing in that none exhibited subnanomolar

potencies, although some had signiﬁcant activities (see Table

1). This observation may be attributed to the polarization of

Scheme 33. Synthesis of Tubulysin Analogues Tb120

a

Reagents and conditions: (a) Et₃N (1.8 equiv), LiCl (1.7 equiv),

PivCl (1.5 equiv), 109 (1.7 equiv), −20 °C, 2 h, 55%; (b) TiCl₄(1.1

equiv), i-Pr₂NEt (1.1 equiv), 1,3,5-trioxane (1.1 equiv), 0 °C, 3.5 h,

61%; (c) NaOMe (1.0 equiv), MeOH, CH₂Cl₂, −78 → 0 °C, 2 h,

94%; (d) 10% Pd/C (50 wt%), H₂, HCl (1.2 equiv; 1 M in MeOH),

MeOH, 23 °C, 0.5 h, 98%; (e) 82 (0.9 equiv), HATU (1.2 equiv),

Et₃N (2.4 equiv), DMF, 0 → 23 °C, 18 h, 94%.

epimeric side products during the stereoselective hydroxyme-

thylation step (110 → 111) as shown in Scheme 33.

2.5. Biological Evaluation of Tubulysin Analogues and

Structure−Activity Relationships. The synthesized ana-

logues were evaluated for their activity against MES SA (uterine

sarcoma cells), MES SA DX (multidrug-resistant uterine

sarcoma cells), and HEK 293T (human embryonic kidney

cancer cells). As shown in Table 1, several of these compounds

exhibited picomolar potencies, with the most notable (high-

lighted with yellow background) being Tb94, Tb95, and

Tb102, and even more potent (highlighted with green

background) Tb64, Tb67, Tb73, Tb93, Tb106, Tb108,

Tb112, Tb117, Tb119, Tb120, and most potent (highlighted

with blue background) Tb107, Tb109, Tb111, Tb115, Tb116,

and Tb118. The latter group (i.e., Tb107, Tb109, Tb111,

Tb115, Tb116, and Tb118) is also notable for their relatively

potent (low nanomolar) cytotoxicities against the marked drug-

resistant cancer cell line MES SA DX.

With the large number of tubulysins synthesized and tested

in our laboratories, and guided by the insights recently obtained

through X-ray crystallographic studies⁵³on tubulin binding

molecules, we were in a position to explain and develop clearer

SARs within the tubulysin family of compounds. The X-ray-

derived structures of N¹⁴-desacetoxytubulysin H (Tb1 in this

study; tubulysin M in ref 53) and its peptide-like relatives HTI-

286⁵³and monomethyl auristatin E (MMAE)⁵³revealed a

binding model that included a number of binding sites on the

tubulysin molecule, including (from “left” to “right”, see Figure

3): (1) basic nitrogen on the “left domain” (protonated form,

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Table 1. Cytotoxicity Data against Cancer Cell Lines MES SA, MES SA DX, and HEK 293T for Tubulysins PTb-D47−PTb-

D49 and Tb50−Tb120

a

IC₅₀= 50% inhibitory concentration of compound against cell growth; MES SA = uterine sarcoma cell line; MES SA DX = MES SA cell line with

b

marked multidrug resistance; HEK 293T = human embryonic kidney cancer cell line; See Supporting Information for further details.

PTb-D43, and DTb-D48), its deprotection to the naked

hydroxyl group (as in Tb58 and Tb60), and its oxidation to the

corresponding ketone (as in Tb59, Tb62, Tb63, and Tb68),

may suggest an unknown structural or biochemical function of

this moiety (e.g., facilitating entrance of the molecule into the

cell).

The thiazole component of Tuv forms two H-bonding

interactions emanating from the thiazole nitrogen atom and the

adjacent carbonyl to the backbone of the β1 tubulin subunit,

thus stabilizing the overall conformation of the central region of

the bound tubulysin molecule. Consequently, any aromatic

Figure 3. Binding interactions of N¹⁴-desacetoxytubulysin H [Tb1,

(tubulysin M)^53a] as deciphered from X-ray crystallographic

analysis.^53a

functionality that maintains these interactions should be

tolerated as long as it does not contribute to additional steric

or electronic constraints within the binding channel, as

demonstrated by analogues Tb64 and Tb65 (pyridine instead

of thiazole). The 5-position (i.e., H-substituted position) of the

thiazole ring is oriented toward an open space⁵³and away from

the interface of the two tubulin monomers, although some

steric constraints could be imposed by substituents in the near

neighborhood of the thiazole ring.⁵³The recognition of this

open space inspires and provides guidance for further

reﬁnement of the tubulysin molecule as potential payloads for

ADCs. Thus, methyl substitution at the 5-position in analogues

Tb66 and Tb67 results in some loss of potency, whereas the

presence of an isopropyl group in analogues Tb72 and Tb73

led to signiﬁcant loss of potency. In contrast, the longer linear

chains, as in Tb110−Tb113, proved beneﬁcial as demonstrated

by their generally increased cytotoxicity potency.

the bonds within these moieties that contribute negatively to

their ﬁtting into the hydrophobic pocket of the receptor.

A more complex explanation may be necessary for the eﬀect

of the spiro cyclopentyl moiety within the isoleucine residue

leading to the lower potency of Tb78, the latter structural motif

most likely changing the overall tertiary conformation of the

tubulysin molecule, thereby decreasing its complementarity to

its binding pocket.

According to the crystallographic analysis,⁵³the acetate

group of the Tuv fragment sits at a narrow channel within the

interface of the α and β tubulin subunits, with no recognizable

H-bonding interactions. The signiﬁcant reduction in potency

that accompanies the removal of this acetate (as in PTb-D42,

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The tubulysin phenylalanine (Tup) domain positioned as it

is at the “right end” of the molecule binds, according to the X-

ray data,⁵³at the edge of the binding channel of tubulin. As

such, it is free to rotate as long as structural changes do not

disturb other binding interactions. Thus, the [1.1.1]-

bicyclopentane containing analogues Tb100, Tb102, and

Tb103 and the cyclohexyl carrying analogue Tb52 are

marginally tolerated, with Tb102 showing subnanomolar

potencies against two of the cell lines tested (see Table 1).

The bulkier naphthalene substituent at this position, as in

Tb104, is also barely tolerated as concluded from its modest

potency compared to the most active compounds (see Table

1). Although para-substitution of the aromatic ring of the Tup

phenylalanine residue is tolerated as evidenced by a number of

active natural tubulysins (e.g., A−C, G, and I)¹⁵⁻¹⁸that contain

a phenolic moiety at that position, the presence of a ﬂuorine

residue in this aromatic ring, as in analogue Tb101, is not,

leading to loss of activity (see Table 1). These data suggest the

importance of a potential π−π interaction of this moiety with a

binding site in the receptor.

The Tup carboxylic acid moiety is involved in a very

important interaction with Arg278 of the tubulin receptor,

forming a salt bridge that provides additional stabilization of the

ligand−receptor complex. The adjacent methyl group appears

to be in an open space region not limited by any apparent steric

or electronic constraints. Removal of this methyl group, as in

Tb105, results in considerable loss of activity (see Table 1).

Most importantly, however, the replacement of this methyl

group of the Tup residue with nitrogen-containing substituents,

as in Tb115−Tb118, translates into high potencies, with

Tb117 and Tb118 being the most impressive.

synthesized compounds led to the identiﬁcation of extremely

potent tubulysin analogues (e.g., Tb107, Tb111, Tb115, and

Tb116, see Table 1 and Figure 2) equipped with hydroxyl,

amino, and carboxylate handles for conjugation to appropriate

targeting systems such as antibodies^50,51and other delivery

systems.⁵²Such conjugates are highly desirable for personalized

targeted chemotherapies, a currently intensely pursued and

rapidly emerging paradigm for cancer treatment. The newly

developed structure−activity relationships described herein are

in line with recently reported X-ray crystallographic analysis

results obtained from a tubulysin−tubulin complex,⁵³and

provide strong pathpointing guidance for further optimization

of the latest tubulysin analogues.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/jacs.7b12692.

■

S

Experimental procedures and characterization data for all

compounds; biological evaluation and data, HEK 293T,

MES SA, and MES SA DX (AbbVie Stemcentrx) (PDF)

AUTHOR INFORMATION

Corresponding Author

*kcn@rice.edu

■

ORCID

K. C. Nicolaou: 0000-0001-5332-2511

Notes

The authors declare no competing ﬁnancial interest.

Figure 4 summarizes the conclusions drawn from these

studies on the eﬀect of structural changes within the four

ACKNOWLEDGMENTS

■

This work was supported by the Cancer Prevention & Research

Institute of Texas (CPRIT), the Welch Foundation (grant C-

1819), and AbbVie Stemcentrx. We thank Drs. Lawrence B.

Alemany and Quinn Kleerekoper (Rice University) for NMR

spectroscopic assistance and Dr. Christopher L. Pennington

(Rice University) for mass spectrometric assistance.

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Improved Total Synthesis of Tubulysins and Design, Synthesis, and Biological Evaluation of New Tubulysins with Highly Potent Cytotoxicities against Cancer Cells as Potential Payloads for Antibody-Drug Conjugates

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