Design, synthesis, and antitumor activity evaluation of pretubulysin analogs

Article abstract of DOI:10.1111/cbdd.13852

Pretubulysin, a biosynthetic precursor of the tubulysins, shows potent biological activity in a variety of tumor cell lines. Although there are several total synthesis routes to tubulysin and pretubulysin reported, the commercialization still has been hampered due to the complexity of the structure. To find structurally simpler pretubulysin analogs, a series of 2-(3-(methylamino)propyl)thiazole-4-carboxamides are designed and synthesized, and their anticancer activities are screened using MCF-7 (breast cancer), and NCI-H157 (lung cancer) cell lines. Taxol (IC₅₀?=?0.01?μM) and pretubulysin are used as the control. Compounds 8c (IC₅₀?=?0.05?μM, MCF-7; 0.09?μM, NCI-H157) and 8h (IC₅₀?=?0.01?μM, MCF-7; 0.02?μM, NCI-H157) exhibited certain antitumor activities comparable to those of Taxol. The urea analogs of pretubulysin might represent a promising scaffold for the further development of novel antitumor drugs.

Full text of DOI:10.1111/cbdd.13852

Article type
: Research Article
Design, synthesis and antitumor activity evaluation of pretubulysin analogs
Xiangrong XU a, Meixia FAN a, Junhui QI a, Lei YAO a,*
(School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University),
Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs
in Universities of Shandong, Yantai University, Yantai 264003, China)
Running title: Pretubulysin analogs
*Corresponding author. E-mail address: yaoleiytu@163.com
School of Pharmacy
Yantai University
30 Qingquan Road, Yantai, Shandong, China, 264005
Tel: +86-535-6948751, Fax:+86-535-6706066
Abstract Pretubulysin, a biosynthetic precursor of the tubulysins, shows potent biological activity
in a variety of tumor cell lines. Although there are several total synthesis routes to tubulysin and
pretubulysin reported, the commercialization still has been hampered due to the complexity of the
structure. To find structurally simpler pretubulysin analogs, a series of
2-(3-(methylamino)propyl)thiazole-4-carboxamides are designed and synthesized, and their
anticancer activities are screened using MCF-7 (breast cancer), and NCI-H157 (lung cancer) cell
lines. Taxol (IC50 = 0.01µM) and pretubulysin are used as the control. Compounds 8c (IC50 = 0.05
µM, MCF-7; 0.09 µM, NCI-H157) and 8h (IC50 = 0.01 µM, MCF-7; 0.02 µM, NCI-H157)
exhibited certain antitumor activities comparable to those of Taxol. The urea analogs of
This article has been accepted for publication and undergone full peer review but has not been
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differences between this version and the Version of Record. Please cite this article as doi:
10.1111/CBDD.13852
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pretubulysin might represent a promising scaffold for the further development of novel antitumor
drugs.
Key words：antitumor activity; pretubulysin; structural modification; tubulysin; urea
1. Introduction
Tubulysins, a family of tetrapeptides isolated from extracts of myxobacteria cultures, showed
extraordinary antitumor activities by binding to tubulin near the vinca alkaloid binding site and
inhibiting tubulin polymerization.1 Even its structure was simpler than tubulysins, pretubulysin (1,
Fig. 1), a presumed biosynthetic precursor of the tubulysins,2-4 was still bearing similar antitumor
efficacy in the nanomolar region.5 Recently, Pretubulysin also displayed potential use in the
treatment for tumor growth,6-8 metastasis,9 and angiogenesis reducing.2 However, the structural
complexity and low yield of pretubulysin led to the commercial supply a challenging issue. Since
the first total synthesis of Tubulysin D reported by Ellman,10 numerous medicinal chemists had put
more effort into pursuing concise total synthesis routes,11,12 and simpler structural analogs.13,14
It was reported that the isopropyl group of the Tuv moiety played an essential role in maintaining
the antitumor activity of pretubulysin.15,16 Compounds would lose antitumor activity when the
isopropyl group was replaced by an aromatic cyclic group or a piperidine group.17,18 Meanwhile,
The Mep and Tup parts were tolerant of many structural modifications.13,19,20 In our lab, we
previously found that the compounds with urea group in Mep part would still possess certain
antitumor activity.18, 21 For example, Compounds 2 and 3 (Fig. 1) exhibited antiproliferative
activity against MCF-7 human breast cell lines with an IC50 value of 0.2 µM and 0.47 µM,
respectively. To simplify the pretubulysin synthesis and increase the hydrophobicity by
introducing aromatic ring(s), a series of 2-(3-(methylamino)propyl)thiazole-4-carboxamides (I,
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Fig.1), which combines both the urea moiety and Tuv fragment of pretubulysin, were designed
and synthesized, and the in vitro antitumor activity was measured by MTT assay.
2. Results and discussion
2.1 Chemistry
The synthesis of 2-(3-(methylamino)propyl)thiazole-4-carboxamides was depicted in Scheme 1. A
three-step synthesis process was employed to transform commercially available ethyl
bromopyruvate 4 into thiazole ester compound 6. Firstly, a thiazole ring was formed by a
cyclization reaction of 4 with thioacetamide in ethanol. Secondly, bromination of the methyl group
afforded the compound ethyl 2-(bromomethyl)thiazole-4-carboxylate 5, by the bromine radical
generated from NBS and dibenzoyl peroxide (BPO). Finally, after converting compound 5 to the
corresponding phosphonium bromide by treatment with triphenylphosphine, then a Witting
reaction with (S)-N-tert-Butoxycarbonyl-N-methylvalinal22afforded the thiazole ester 6 in
moderate yield. The olefin group of intermediate 6 was reduced under hydrogen conditions, and
the ester group was then hydrolyzed under alkaline conditions to afford the corresponding
carboxylic acid. The resultant acid and a variety of amines, including methylamine, benzylamine,
and phenylethylamine, yielded amides 7a–7c under classic peptide coupling condition (HOBt,
EDCI, DIPEA). Removal of the Boc group in compounds 7a–7c, followed by coupling with a
series of 2-ureidoacetic acid (prepared from L-valine or L-leucine with isocyanates)23 afforded 8a–
8q in moderate yields.
2.2 Cell viability assay
The structures of all compounds and their antitumor activities were listed in Table 1. This series
of compounds only had 2 or 3 stereocenters compared to pretubulysin which had 6. Although the
antitumor activities of these compounds were discovered to be 10-fold less than that of
pretubulysin, certain compounds exhibited potent antitumor activity comparable to those of Taxol.
Compound 8c showed the antitumor activities, with IC50 values of 0.05 μM against MCF-7 breast
cancer cell, and 0.09 μM against NCI-H157 lung cancer cell. The most potent compound 8h
showed the antitumor activities, with IC50 values of 0.01 μM against MCF-7 breast cancer cells,
and 0.02 μM against NCI-H157lung cancer cells. Previous studies including our lab’s work
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showed that the central Tuv part played a critical role in maintaining the antitumor activity of
tubulysins and pretubulysin.17, 24 Both the right end (Tup part), and the left end (Mep part) were
tolerant of many modifications. When comparing the three R1 groups of these analogs (Tup part of
pretubulysin), those compounds containing phenylethylamine fragments generally possessed
better activity than those with methylamine and benzylamine fragments (e.g., 8i vs. 8b and 8a). It
might suggest that the phenyl group in the Tup was important for maintaining the activity. When
comparing the four R3 groups of these analogs (Mep part of pretubulysin), those compounds with
halogen-substituted phenyl group generally possessed better activity. For example, compounds 8b
(IC50 = 1.05 µM, MCF-7) and 8c (IC50 = 0.15 µM, MCF-7) were more potent than compound 8a
(IC50 = 1.75 µM, MCF-7); compounds 8e (IC50 = 0.79 µM, MCF-7) and 8f (IC50 = 0.53 µM,
MCF-7) were more potent than compound 8d (IC50 = 1.27 µM, MCF-7). These phenomena were
often observed in synthesized small-molecule antitumor agents, especially in the receptor tyrosine
kinases (RTK) inhibitors.25,26 The halogen group was recognized as the second favorite
heteroatom in drug design, and a large number of halogen-containing drugs were approved by
FDA or under investigation.27,28 Regarding the R2 group, it seemed that the leucine analogs were
much better than valine ones in most cases, and at least the similar effect in some cases. For
example, Compound 8c (IC50 = 0.05 µM) were more potent than compound 8m (IC50 = 0.76 µM)
against MCF-7 and NCI-H157 cancer cells, and compound 8h (IC50 = 0.01 µM) was more potent
than compound 8p (IC50 = 0.38 µM). Compounds 8n (IC50 = 1.02 µM, MCF-7) and 8d (IC50 =
1.27 µM, MCF-7) had similar antitumor activity; compounds 8j (IC50 = 0.09 µM) and 8q (IC50 =
0.11 µM) exhibited almost the same antitumor activity against MCF-7 breast cancer cells.
Although these urea analogs were less potent than that of pretubulysin, compound 8h exhibited
potent antitumor activity comparable to that of Taxol. Also, these compounds were more potent
than our previously prepared Tuv aromatic or cyclization analogs.17 The lipophilicity as calculated
by the ClogP (4.4-8.3) of these compounds was higher than that of pretubulysin (3.8). There were
only 2 or 3 stereocenters in these structures, and it was easier to obtain from commercial starting
materials. The stereocenters decrease meant lots of saving on the studies on the stereoisomers and
related substances.
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2.3 Molecular docking
To better understand how these compounds interact with tubulin, molecular docking study was
performed using the co-crystal structure of microtubules and tubulysin M (PDB code: 4ZOL) by
Sybyl 2.1.1 software.29 Pretubulysin, compound 8c, and compound 8h were chosen for the
molecular docking study, according to the general protocol. The predicted binding mode was
shown in Fig. 2.
For pretubulysin, the two carbonyl groups of Tup and Mep had hydrogen bond interaction with the
amino acid residue Tyr224, Lys 352, and Val 353, with the total scores of 9.8140 (A, Fig. 2).
However, compounds 8c and 8h adopted different conformers to interact with the tubulin. As for
compound 8c, the urea group had hydrogen bond interaction with Asp179 and Asn329.
Meanwhile, the trifluoromethyl group had additional interaction with the Ala333, with the total
scores of 9.2288 (B, Fig. 2). These phenomena might partially explain that compound 8c had a
better antitumor potency, even with a methyl group in the Tup part. As for compound 8h, the
Asn329 amino acid had interactions with the urea group and carbonyl group, with total scores of
9.1030. Both compounds 8c and 8h lacked the hydrogen bond interaction of Tyr224 with the
carbonyl group of Tup, which might be the reasons for the reducing potency of antitumor activity
of these urea analogs.
3. Conclusion
In summary, a number of new 2-(3-(methylamino)propyl)thiazole-4-carboxamides were
synthesized, and their anticancer activities were evaluated by MTT method using MCF-7-breast
cancer and NCI-H157-lung cancer cell lines. Compound 8c showed the antitumor activities, with
IC50 values of 0.05 μM against MCF-7 breast cancer cells, and 0.09 μM against NCI-H157 lung
cancer cells. The most potent compound 8h showed the antitumor activities, with IC50 values of
0.01 μM against MCF-7 breast cancer cell, and 0.02 μM against NCI-H157 lung cancer cell.
Although the antitumor activities of these urea compounds were less potent than that of the
pretubulysin, compound 8h showed potent antitumor activity comparable to that of Taxol. It was
relatively easier to synthesize this series of more lipophilic compounds to enhance oral absorption.
This scaffold might represent a promising lead for the further development of novel antitumor
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drugs.
4. Experimental Section
4.1 Chemistry
General
Unless otherwise stated, all reactions were performed in flame-dried glassware equipped with
glass stoppers under positive pressure of Ar with magnetic stirring. The NMR spectra of the
intermediates and final products in deuterated chloroform were detected on a Bruker 400 or 600
MHz spectrometer (see Supporting Information). High-resolution mass spectra (HRMS) were
recorded on an Agilent 6210 ESI/TOF mass spectrometer. Melting points were determined using a
digitizing melting point apparatus (WRS-1B, Shanghai Precision & Scientific Instrument Co.,
Ltd.) and were uncorrected. All synthesized compounds were purified by column chromatography
on silica gel from Qingdao Ocean Chemical Co. (200 to 300 mesh) with particle size from 54 to
74 μm using ethyl acetate and hexane (or petroleum ether) as the eluent. Analytical TLC was
carried out on Merck pre-coated silica gel 60 GF-254 using 0.25-mm-thick TLC plates. CLogP
was obtained by ChemBioDraw Ultra 12.0 software.
General procedure for compound 8
2-((R)-3-((S)-2-(3-(4-Methoxylphenyl)ureido)-N,4-dimethylpentanamido)-4-methylpentyl)-N-me
thylthiazole-4-carboxamide (8a).
Step 1: To a solution of 7a (2.35 g, 6.6 mmol) in DCM (30 mL), trifluoroacetic acid (TFA, 15 mL)
was added at room temperature. The reaction mixture was stirred at room temperature for 3 h.
Then the mixture was concentrated under reduced pressure, and the obtained residue was washed
with water. The aqueous solution was cautiously neutralized with excess, solid NaHCO3 and
extracted with ethyl acetate (3×30 mL). The extracted organic layers were dried over anhydrous
Na2SO4, filtered, concentrated to yield (1.48 g, 87%) the amine intermediated as a yellow oil and
used in the next step directly.
Step 2: (3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.14 g, 0.72 mmol) and
1-hydroxybenzotriazole (0.09 g, 0.60 mmol) was added to
a
solution of
((4-methoxyphenyl)carbamoyl)-L-leucine (0.13 g, 0.44 mmol) in DMF (2 mL). After the mixture
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was stirred at room temperature for 30 min, the above amine intermediate (0.1 g, 0.4 mmol) was
added. Then the reaction mixture was stirred at room temperature for 6 h before water (6 mL) was
added to quench the reaction. The aqueous solution was extracted with ethyl acetate (3×15 mL),
and the extracted organic layers were dried over anhydrous Na2SO4, filtered, and concentrated.
The residue was purified by flash chromatography to yield 8a (0.08 g, 40%) as a yellow solid. mp
83.3-85.2 °C; 1H NMR (400 MHz, CDCl3): δ 7.82 (s, 1H, CH), 7.60 (br s, 1H, NH), 7.32-7.31 (m,
1H, NH), 7.02 (d, J = 8 Hz, 2H, ArH), 6.74-6.68 (m, 1H, ArH), 6.53 (d, J =8 Hz, 2H, ArH),
4.90-4.86 (m, 1H, CH), 4.34-4.30 (m, 1H, CH), 3.63 (s, 3H, OCH3), 3.00 (s, 3H, NCH3), 2.94-2.91
(m, 1H, CH), 2.89 (s, 3H, CH3), 2.75-2.71 (m, 1H, CH), 2.51-2.49 (m, 1H, CH), 2.10-2.03 (m, 1H,
CH), 1.83-1.79 (m, 2H, CH2), 1.71-1.67 (m, 1H, CH), 1.54-1.50 (m, 1H, CH), 1.44-1.40 (m, 1H,
CH), 0.98 (d, J = 8.0 Hz, 3H, CH3), 0.94 (d, J = 8.0 Hz, 6H, 2XCH3), 0.80 (d, J = 8.0 Hz, 3H,
CH3); 13C NMR (101 MHz, CDCl3): δ 176.5, 170.2, 161.9, 156.4, 155.3, 149.5, 132.0, 122.3,
121.8, 113.8, 55.3, 49.0, 41.6, 30.2, 30.0, 29.3, 26.0, 24.8, 23.4, 21.8, 20.1, 19.8. HRMS (m/z):
[M+H]+ calcd for C26H39N5O4S: 517.2723; found: 517.2728.
2-((R)-3-((S)-2-(3-(4-Chlorophenyl)ureido)-N,4-dimethylpentanamido)-4-methylpentyl)-N-met
hylthiazole-4-carboxamide (8b)
Prepared from 7a by the same method as described in 8a. White solid (0.07 g, 35%); mp
82.4-84.5 °C; 1H NMR (400 MHz, CDCl3): δ 7.88 (s, 1H, CH), 7.78-7.74 (m, 1H, NH), 7.28-7.25
(m, 1H, NH), 7.03 (d, J = 8.0 Hz, 2H, ArH), 6.99-6.97 (m, 1H, NH), 6.91 (d, J = 8.0 Hz, 2H,
ArH), 4.88-4.84 (m, 1H, CH), 4.36-4.28 (m, 1H, CH), 3.02 (s, 3H, NCH3), 2.95-2.94 (m, 1H, CH),
2.92 (s, 3H, CH3), 2.80-2.77 (m, 1H, CH), 2.30-2.27 (m, 1H, CH), 2.17-2.10 (m, 1H, CH),
1.89-1.79 (m, 2H, CH2), 1.57-1.51 (m, 1H, CH), 1.45-1.39 (m, 1H, CH), 0.99 (d, J = 8.0 Hz, 6H,
13
2XCH3), 0.92 (d, J = 8.0 Hz, 3H, CH3), 0.82 (d, J = 8.0 Hz, 3H, CH3); C NMR (100 MHz,
CDCl3): δ 176.8, 170.1, 161.9, 155.8, 149.6, 137.7, 128.3, 127.1, 122.4, 120.3, 49.3, 41.2, 30.2,
29.9, 29.1, 26.1, 24.9, 23.5, 21.6, 20.2, 19.8; HRMS (m/z): [M+H]+ calcd for C25H36ClN5O3S:
521.2227; found: 521.2230.
2-((R)-3-((S)-2-(3-(4-Chloro-3-(trifluoromethyl)phenyl)ureido)-N,4-dimethylpentanamido)-4-
methylpentyl)-N-methylthiazole-4-carboxamide (8c).
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Prepared from 7a by the same method as described in 8a. Yellow solid, (0.08 g, 33.3%); mp
1
81.3-83.5 °C; H NMR (400 MHz, CDCl3): δ 7.99 (s, 1H, CH), 7.91 (s, 1H, NH), 7.38 (d, J = 4
Hz, 1H, NH), 7.28-7.25 (m, 1H, NH), 7.23-7.21 (m, 1H, ArH), 7.20-7.19 (m, 1H, ArH), 7.02-6.99
(m, 1H, ArH), 4.84-4.79 (m, 1H, CH), 4.40-4.32 (m, 1H, CH), 3.04 (s, 3H, NCH3), 2.98-2.95 (m,
1H, CH), 2.93 (s, 3H, CH3), 2.86-2.84 (m, 1H, CH), 2.82-2.78 (m, 1H, CH), 2.22-2.14 (m, 1H,
CH), 1.92-1.87 (m, 1H, CH), 1.57-1.53 (m, 1H, CH), 1.49-1.40 (m, 1H, CH), 1.24-1.23 (m, 1H,
CH), 1.0 (d, J = 8 Hz, 6H, 2XCH3), 0.95 (d, J =8.0 Hz, 3H, CH3), 0.83 (d, J = 8.0 Hz, 3H, CH3);
13C NMR (100 MHz, CDCl3): δ 176.9, 169.9, 169.6, 161.8, 161.7, 155.5, 155.4, 149.9, 149.7,
138.0, 131.3, 122.4 (CF3), 117.5, 49.6, 40.9, 30.1, 29.2, 26.0, 24.9, 23.5, 21.3, 20.1, 19.7; HRMS
(m/z): [M+H]+ calcd for C26H35ClF3N5O3S: 589.2101; found: 589.2105.
N-Benzyl-2-((R)-3-((S)-2-(3-(4-methoxyphenyl)ureido)-N,4-dimethylpentanamido)-4-methylpe
ntyl)thiazole-4-carboxamide (8d).
Prepared from 7b by the same method as described in 8a. Yellow solid, (0.06 g, 21%); mp
65.5-66.8 °C; 1H NMR (400 MHz, CDCl3): δ 7.89 (s, 1H, CH), 7.72-7.65 (m, 1H, NH), 7.35-7.29
(multiple, 5H, ArH), 7.27-7.25 (m, 1H, ArH), 7.20-7.14 (m, 1H, ArH), 7.04 (d, J = 8 Hz,, 2H,
ArH), 6.59 (d, J = 4 Hz, 1H, ArH), 6.44-6.40 (m, 1H, CH), 4.91-4.85 (m, 1H, CH), 4.59 (d, J = 4
Hz,, 1H, CH), 4.34-4.30 (m, 1H, CH), 3.72-3.70 (m, 1H, CH), 3.67-3.65 (s, 3H, OCH3), 2.99 (s,
3H, NCH3), 2.92-2.84 (m, 1H, CH), 2.75-2.68 (m, 1H, CH), 2.12-2.03 (m, 1H, CH), 1.84-1.79 (m,
2H, CH2), 1.72-1.64 (m, 1H, CH), 1.56-1.49 (m, 1H, CH), 1.43-1.38 (m, 1H, CH), 1.28-1.23 (m,
1H, CH), 0.9 9 (d, J = 8.0 Hz, 3H, CH3), 0.94 (d, J = 8.0 Hz, 6H, 2xCH3), 0.81 (d, J = 8.0 Hz, 3H,
CH3); 13C NMR (100 MHz, CDCl3): δ 176.5, 170.2, 161.2, 156.4, 155.4, 149.5, 138.5, 132.0,
128.7, 127.9, 127.4, 122.8, 122.0, 113.9, 55.4, 49.1, 43.2, 41.7, 30.2, 30.0, 29.2, 24.8, 23.5, 21.8,
20.1, 19.8; HRMS (m/z): [M+H]+ calcd for C32H43N5O4S: 593.3036; found: 593.3039.
N-Benzyl-2-((R)-3-((S)-2-(3-(4-chlorophenyl)ureido)-N,4-dimethylpentanamido)-4-methylpen
tyl)thiazole-4-carboxamide (8e).
Prepared from 7b by the same method as described in 8a. Yellow solid, (0.07 g, 23%); mp
70.2-72.5 °C; 1H NMR (400 MHz, CDCl3): δ 7.94 (s, 1H, CH), 7.55-7.52 (m, 1H, NH), 7.35-7.31
(m, 5H, ArH), 7.27-7.23 (m, 1H, NH), 7.0 (d, J = 8.9 Hz, 2H, ArH), 6.90 (d, J = 8.9 Hz, 2H, ArH),
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5.31-5.23 (m, 1H, NH), 4.6 (t, J = 5.8 Hz, 2H, CH2), 4.37-4.28 (m, 1H, CH), 3.01 (s, 3H, NCH3),
2.95-2.91 (m, 1H, CH), 2.80-2.74 (m, 1H, CH), 2.17-2.10 (m, 1H, CH), 1.88-1.83 (m, 1H, CH),
1.76-1.72 (m, 1H, CH), 1.56-1.53 (m, 1H, CH), 1.46-1.39 (m, 1H, CH), 1.26-1.22 (m, 1H, CH),
1.0 (d, J = 8.0 Hz, 6H, 2XCH3), 0.92 (d, J = 8.0 Hz, 3H, CH3), 0.82 (d, J = 6.6 Hz, 3H, CH3); 13C
NMR (100 MHz, CDCl3): δ 176.8, 170.1, 169.8, 161.1, 155.8, 149.5, 138.4, 137.6, 128.7, 128.4,
127.9, 127.4, 123.0, 120.3, 49.3, 43.3, 41.2, 30.2, 29.7, 24.91, 23.5, 21.6, 20.2, 19.8; HRMS (m/z):
[M+H]+ calcd for C31H40ClN5O3S: 597.2540; found: 597.2544.
N-Benzyl-2-((R)-3-((S)-2-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)-N,4-dimethylpentan
amido)-4-methylpentyl)thiazole-4-carboxamide (8f).
Prepared from 7b by the same method as described in 8a. Yellow solid, (0.06 g, 21%); mp
1
147.5-149.3 °C; H NMR (400 MHz, CDCl3): δ 7.96 (s, 1H, CH), 7.79-7.75 (m, 1H, NH),
7.65-7.61 (m, 1H, NH), 7.33-7.31 (m, 5H, ArH), 7.25-7.24 (m, 1H, ArH), 7.20-7.17 (m, 1H, ArH),
7.12-7.10 (m, 1H, ArH), 6.98-6.96 (m, 1H, NH), 4.81-4.76 (m, 1H, CH), 4.61 (d, J = 1.9 Hz, 1H,
CH), 4.37 (d, J = 8.6 Hz,, 1H, CH), 3.63-3.60 (m, 1H, CH), 3.02 (s, 3H, NCH3), 2.98-2.94 (m, 1H,
CH), 2.87-2.83 (m, 1H, CH), 2.76 (dd, J = 10.3, 5.1 Hz,, 1H, CH), 2.19-2.12 (m, 1H, CH),
1.88-1.82 (m, 1H, CH), 1.52 (dd, J = 14.3, 3.9 Hz, 1H, CH), 1.50-1.40 (m, 1H, CH), 1.24 (d, J =
1.2 Hz, 1H, CH), 0.99 (d, J = 6.8 Hz, 3H, CH3), 0.93 (d, J = 6.8 Hz, 3H, CH3), 0.89 (d, J = 9.2 Hz,
13
3H, CH3), 0.82 (d, J = 6.5 Hz 3H, CH3); C NMR (100 MHz, CDCl3): δ 176.8, 169.9, 169.7,
161.1, 161.0, 155.5, 149.6, 138.3, 137.9, 131.3, 128.8, 128.7, 127.9, 127.5, 123.0 (CF3), 49.7,
43.3, 40.9, 30.2, 29.2, 24.9, 23.5, 21.3, 20.2, 19.7; HRMS (m/z): [M+H]+ calcd for
C32H39ClF3N5O3S: 665.2414; found: 665.2417.
2-((R)-3-((S)-N,4-Dimethyl-2-(3-(4-nitrophenyl)ureido)pentanamido)-4-methylpentyl)-N-phen
ethylthiazole-4-carboxamide (8g).
Prepared from 7c by the same method as described in 8a. Yellow solid, (0.12 g, 57%); mp
73.4-75.6 °C; 1H NMR (400 MHz, CDCl3): δ 8.14-8.11 (m, 1H, CH), 7.96 (s, 1H, NH), 7.85-7.82
(m, 2H, ArH), 7.43-7.33 (m, 1H, NH), 7.29-7.27 (m, 1H, ArH), 7.25-7.22 (m, 2H, ArH), 7.21-7.18
(m, 3H, ArH), 7.11-7.09 (m, 1H, ArH), 4.85-4.80 (m, 1H, CH), 4.41-4.30 (m, 1H, CH), 3.71-3.64
(m, 2H, CH2), 3.04 (s, 3H, NCH3), 2.93-2.90 (m, 1H, CH), 2.87 (d, J = 8.1 Hz, 1H, CH), 2.80-2.78
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(m, 1H, CH), 2.24-2.17 (m, 1H, CH), 1.88-1.82 (m, 1H, CH), 1.67 (d, J = 12.9 Hz, 1H, CH), 1.53
(dd, J = 18.4, 7.7 Hz, 1H, CH), 1.34 (d, J = 14.6 Hz, 1H, CH), 1.27-1.24 (m, 1H, CH), 1.03 (d, J =
8.0 Hz, 6H, 2XCH3), 0.95 (d, J = 8.0 Hz, 3H, CH3), 0.85 (d, J = 6.6 Hz, 3H, CH3); 13C NMR (100
MHz,CDCl3): δ 169.9, 161.1, 155.7, 149.7, 139.0, 137.6, 128.9, 128.6, 128.4, 127.2, 126.4, 122.5,
120.4, 49.3, 41.3, 40.7, 35.9, 30.2, 29.7, 26.5, 24.9, 23.5, 21.6, 20.2, 19.8; HRMS (m/z): [M+H]+
calcd for C32H42N6O5S: 622.2937; found: 622.2941.
2-((R)-3-((S)-2-(3-(4-Chlorophenyl)ureido)-N,4-dimethylpentanamido)-4-methylpentyl)-N-phe
nethylthiazole-4-carboxamide (8h).
Prepared from 7c by the same method as described in 8a. Yellow solid, (0.10 g, 47%); mp
78.8-80.3 °C; 1H NMR (400 MHz, CDCl3): δ 7.89 (s, 1H, CH), 7.60-7.58 (m, 1H, NH), 7.38-7.35
(m, 1H, NH), 7.28-7.26 (m, 1H, NH), 7.25-7.19 (m, 5H, ArH), 7.02 (d, J = 8.9 Hz, 2H, ArH), 6.93
(d, J = 8.9 Hz, 2H, ArH), 4.86-4.84 (m, 1H, CH), 3.69-3.62 (m, 2H, CH2), 3.03 (s, 3H, NCH3),
2.89 (t, J = 7.3 Hz, 2H, CH2), 2.81-2.72 (m, 2H, CH2), 2.18-2.12 (m, 1H, CH), 2.10-2.03 (m, 1H,
CH), 1.87-1.86 (m, 1H, CH), 1.78-1.74 (m, 1H, CH), 1.58-1.51 (m, 1H, CH), 1.47-1.41 (m, 1H,
CH), 1.26-1.23 (m, 1H, CH), 1.02 (d, J = 6.7 Hz, 3H, CH3), 0.98 (d, J = 12.0 Hz, 3H, CH3), 0.94
(d, J = 12.0 Hz, 3H, CH3), 0.84 (d, J = 6.6 Hz, 3H, CH3); 13C NMR (100 MHz,CDCl3): δ 169.9,
161.1, 155.8, 155.6, 149.7, 139.0, 137.6, 128.9, 128.6, 128.4, 127.2, 126.4, 122.6, 120.3, 49.4,
40.7, 40.6, 35.9, 30.2, 29.7, 24.9, 23.5, 21.5, 21.4, 20.2, 19.8; HRMS (m/z): [M+H]+ calcd for
C32H42ClN5O3S: 611.2697; found: 611.2702.
2-((R)-3-((S)-2-(3-(4-Methoxyphenyl)ureido)-N,4-dimethylpentanamido)-4-methylpentyl)-N-p
henethylthiazole-4-carboxamide (8i).
Prepared from 7c by the same method as described in 8a. Yellow solid, (0.08 g, 38%); mp
67.3-69.7 °C; 1H NMR (400 MHz, CDCl3): δ 7.85 (s, 1H, CH), 7.44-7.42 (m, 1H, NH), 7.30-7.27
(m, 3H, ArH), 7.23-7.17 (m, 4H, ArH), 7.07 (d, J = 6.0 Hz, 2H, ArH), 6.62 (d, J = 6.0 Hz, 2H,
ArH), 4.91-4.88 (m, 1H, CH), 4.36-4.33 (m, 1H, CH), 3.71-3.69 (m, 1H, CH), 3.68 (s, 3H, OCH3),
3.63-3.61 (m, 1H, CH), 3.00 (s, 3H, NCH3), 2.91-2.87 (m, 2H, CH2), 2.75 (dd, J = 12.6, 4.8 Hz,
1H, CH), 2.13-2.06 (m, 1H, CH), 1.86-1.81 (m, 2H, CH2), 1.74-1.70 (m, 1H, CH), 1.57-1.51 (m,
1H, CH), 1.43 (t, J = 9.5 Hz, 1H, CH), 1.25-1.23 (m, 1H, CH), 1.02 (d, J = 6.5 Hz, 3H, CH3),
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0.98 (d, J = 8.0 Hz, 3H, CH3), 0.92 (d, J = 8.0 Hz, 3H, CH3), 0.83 (d, J = 6.6 Hz, 3H, CH3); 13C
NMR (100 MHz, CDCl3): δ 176.5, 170.1, 161.2, 156.4, 155.5, 149.6, 139.0, 131.9, 128.8, 128.6,
126.4, 122.5, 122.1, 113.9, 55.4, 49.13, 41.7, 40.7, 35.9, 30.2, 29.7, 24.9, 23.5, 21.8, 20.2, 19.9;
HRMS (m/z): [M+H]+ calcd for C33H45N5O4S: 607.3192; found: 607.3197.
2-((R)-3-((S)-2-(3-(4-Chloro-3-(trifluoromethyl)phenyl)ureido)-N,4-dimethylpentanamido)-4-
methylpentyl)-N-phenethylthiazole-4-carboxamide (8j).
Prepared from 7c by the same method as described in 8a. Yellow solid, (0.07 g, 36%); mp
1
151.3-153.2 °C; H NMR (400 MHz, CDCl3): δ 7.92 (s, 1H, CH), 7.87-7.81 (m, 1H, NH),
7.37-7.34 (m, 1H, NH), 7.31-7.28 (m, 1H, ArH), 7.27-7.25 (m, 2H, ArH), 7.22-7.15 (m, 5H, ArH),
7.0 (d, J = 8.6 Hz, 1H, NH), 4.83-4.78 (m, 1H, CH), 4.39-4.35 (m, 1H, CH), 3.67 (d, J = 7.1 Hz,
1H, CH), 3.03 (s, 3H, NCH3), 2.99-2.97 (m, 1H, CH), 2.94-2.92 (m, 1H, CH), 2.91-2.89 (m, 1H,
CH), 2.87-2.85 (m, 1H, CH), 2.81-2.73 (m, 1H, CH), 2.20-2.15 (m, 1H, CH), 1.89-1.85 (m, 2H,
CH2), 1.53 (dd, J = 16.2, 12.5 Hz, 1H, CH), 1.49-1.41 (m, 1H, CH), 1.23 (d, J = 7.1 Hz, 1H,
CH), 1.02 (d, J = 6.5 Hz, 3H, CH3), 0.97 (d, J = 8.0 Hz, 3H, CH3), 0.94 (d, J = 8.0 Hz, 3H, CH3),
0.84 (d, J = 6.5 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3): δ 176.9, 169.8, 169.6, 161.2, 161.0,
155.5, 155.4, 149.7, 139.0, 138.0, 131.3, 128.8, 128.6, 126.4, 122.6 (CF3), 49.7, 40.7, 40.6, 35.9,
30.3, 30.1, 29.1, 24.9, 23.5, 21.3, 20.1, 19.8; HRMS (m/z): [M+H]+ calcd for C33H41ClF3N5O3S:
679.2571; found: 679.2574.
2-((R)-3-((S)-2-(3-(4-Methoxyphenyl)ureido)-N,3-dimethylbutanamido)-4-methylpentyl)-N-m
ethylthiazole-4-carboxamide (8k).
Prepared from 7a by the same method as described in 8a. Yellow solid, (0.11 g, 55%); mp
73.7-75.9 °C; 1H NMR (400 MHz, CDCl3): δ 7.82 (s, 1H, CH), 7.61-7.56 (m, 1H, NH), 7.36-7.27
(m, 1H, NH), 7.25-7.16 (m, 2H, ArH), 6.81-6.71 (m, 2H, ArH), 6.26-6.24 (m, 1H, NH), 5.27 (s,
1H, CH), 4.75-4.65 (m, 1H, CH), 3.71 (s, 3H, OCH3), 3.04 (s, 3H, NCH3), 2.96-2.95 (m, 1H, CH),
2.91-2.89 (m, 3H, CH3), 2.76-2.57 (m, 1H, CH), 2.09 (dd, J = 16.4, 8.0 Hz, 1H, CH), 2.04-1.99
(m, 1H, CH), 1.71-1.68 (m, 1H, CH), 1.23 (s, 1H, CH), 1.02 (d, J = 4.0 Hz, 3H, CH3), 1.00-0.96
(m, 6H, CH3), 0.85 (d, J = 6.6 Hz 3H, CH3); 13C NMR (100 MHz, CDCl3): δ 175.3, 170.1, 169.7,
161.9, 156.5, 156.3, 149.6, 132.0, 122.3, 114.2, 55.5, 31.2, 30.9, 30.3, 30.1, 26.0, 20.2, 19.9, 19.8,
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18.0; HRMS (m/z): [M+H]+ calcd for C25H37N5O4S: 503.2566; found: 503.2569.
2-((R)-3-((S)-2-(3-(4-Chlorophenyl)ureido)-N,3-dimethylbutanamido)-4-methylpentyl)-N-m
ethylthiazole-4-carboxamide (8l).
Prepared from 7a by the same method as described in 8a. White solid, (0.13 g, 65%); mp
74.5-76.7 °C; 1H NMR (400 MHz, CDCl3): δ 8.01-7.95 (m, 1H, CH), 7.82 (s, 1H, NH), 7.37-7.35
(m, 1H, NH), 7.26 (d, J = 9.0 Hz, 2H, ArH), 7.12 (d, J = 9.0 Hz, 2H, ArH), 6.54-6.49 (m, 1H,
NH), 5.27 (s, 1H, CH), 4.74-4.65 (m, 1H, CH), 3.07 (s, 3H, CH3), 2.97-2.93 (m, 1H, CH), 2.91 (s,
3H, NCH3), 2.78-2.74 (m, 1H, CH), 2.14-2.09 (m, 1H, CH), 2.04 (d, J = 7.7 Hz, 1H, CH),
1.77-1.74 (m, 1H, CH), 1.23 (s, 1H, CH), 1.04-0.99 (m, 9H, CH3), 0.87 (d, J = 6.6 Hz, 3H, CH3);
13C NMR (100 MHz, CDCl3): δ 175.5, 169.9, 161.9, 155.7, 149.6, 138.0, 128.8, 127.3, 122.4,
120.4, 55.6, 53.5, 30.9, 30.3, 30.1, 29.3, 26.0, 20.2, 19.8, 18.1; HRMS (m/z): [M+H]+ calcd for
C24H34ClN5O3S: 507.2071; found: 507.2076.
2-((R)-3-((S)-2-(3-(4-Chloro-3-(trifluoromethyl)phenyl)ureido)-N,3-dimethylbutanamido)-4-
methylpentyl)-N-methylthiazole-4-carboxamide (8m).
Prepared from 7a by the same method as described in 8a. White solid, (0.09 g, 45%); mp
82.3-84.2 °C; 1H NMR (400 MHz, CDCl3): δ 8.01-7.98 (m, 1H, NH), 7.85 (s, 1H, NH), 7.68-7.66
(m, 1H, NH), 7.62-7.59 (m, 1H, ArH), 7.53-7.50 (m, 1H, ArH), 7.29-7.25 (m, 1H, ArH), 6.73-6.55
(m, 1H, NH), 4.77-4.68 (m, 1H, CH), 4.34-4.29 (m, 1H, CH), 3.06 (s, 3H, CH3), 2.98 (d, J = 5.1
Hz, 1H, CH), 2.93 (s, 3H, NCH3), 2.79 (d, J = 4.7 Hz, 1H, CH), 2.20 (dd, J = 11.0, 6.9 Hz, 1H,
CH), 2.06-2.02 (m, 1H, CH), 1.68-1.65 (m, 1H, CH), 1.24 (s, 1H, CH), 1.05 (d, J = 6.4 Hz, 3H,
13
CH3), 1.02 (d, J = 8.0 Hz, 6H, 2XCH3), 0.87 (d, J = 6.5 Hz, 3H, CH3); C NMR (100 MHz,
CDCl3): δ 175.7, 175.2, 169.7, 169.4, 161.8, 155.4, 150.9, 149.6, 138.4, 131.7, 122.8 (CF3), 122.4,
55.7, 30.8, 30.1, 29.7, 26.0, 22.7, 20.2, 19.8, 17.9, 14.1; HRMS (m/z): [M+H]+ calcd for
C25H33ClF3N5O3S: 575.1945; found: 575.1949.
N-Benzyl-2-((R)-3-((S)-2-(3-(4-Methoxyphenyl)ureido)-N,3-dimethylbutanamido)-4-methylpe
ntyl)thiazole-4-carboxamide (8n).
Prepared from 7b by the same method as described in 8a. White solid, (0.06 g, 35%); mp
75.5-77.8 °C; 1H NMR (400 MHz, CDCl3): δ 7.87 (s, 1H, CH), 7.76-7.71 (m, 1H, NH), 7.32-7.28
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(m, 5H, ArH), 7.25-7.23 (m, 1H, NH), 7.12 (d, J = 9.0 Hz, 2H, ArH), 6.70 (d, J = 9.0 Hz, 2H,
ArH), 6.09-6.02 (m, 1H, NH), 4.63-4.53 (m, 1H, CH), 4.31-4.27 (m, 1H, CH), 3.74-3.72 (m, 1H,
CH), 3.70 (s, 3H, OCH3), 2.99 (s, 3H, NCH3), 2.95-2.94 (m, 1H, CH), 2.87-2.85 (m, 1H, CH),
2.71-2.68 (m, 1H, CH), 2.07-2.00 (m, 1H, CH), 1.86-1.85 (m, 1H, CH), 1.70-1.65 (m, 1H, CH),
13
1.24 (s, 1H, CH), 1.01-0.93 (m, 9H, CH3), 0.83 (d, J = 6.6 Hz, 3H, CH3); C NMR (100 MHz,
CDCl3): δ 170.1, 169.8, 161.2, 161.1, 156.4, 155.9, 149.5, 138.5, 131.8, 128.7, 127.7, 127.4,
122.8, 114.2, 55.5, 43.3, 43.2, 30.9, 30.1, 29.7, 29.4, 26.1, 20.2, 19.8, 17.9; HRMS (m/z): [M+H]+
calcd for C31H41N5O4S: 579.2879; found: 579.2883.
2-((R)-3-((S)-2-(3-(4-Methoxyphenyl)ureido)-N,3-dimethylbutanamido)-4-methylpentyl)-N-phe
nethylthiazole-4-carboxamide (8o).
Prepared from 7c by the same method as described in 8a. White solid, (0.08 g, 47%); mp
1
156.2-158.7 °C; H NMR (400 MHz, CDCl3): δ 7.83 (s, 1H, CH), 7.44-7.41 (m, 1H, NH),
7.37-7.33 (m, 1H, NH), 7.29-7.26 (m, 2H, ArH), 7.23-7.20 (m, 3H, ArH), 7.12 (d, J = 9.0 Hz, 2H,
ArH), 6.72 (d, J = 9.0 Hz, 2H, ArH), 6.19-6.12 (m, 1H, NH), 4.75-4.66 (m, 1H, CH), 4.33-4.29
(m, 1H, CH), 3.74-3.72 (m, 1H, CH), 3.71 (s, 3H, OCH3), 3.63-3.60 (m, 1H, CH), 3.02 (s, 3H,
NCH3), 2.94-2.92 (m, 1H, CH), 2.90-2.87 (m, 1H, CH), 2.74 (d, J = 2.2 Hz, 1H, CH), 2.11-1.99
(m, 2H, CH2), 1.87-1.86 (m, 1H, CH), 1.69 (dd, J = 12.9, 6.2 Hz, 1H, CH), 1.24 (s, 1H, CH),
1.02-0.96 (multiple, 9H, 3XCH3), 0.85 (d, J = 6.6 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3): δ
170.0, 169.8, 161.2, 161.1, 156.4, 155.9, 149.6, 139.0, 131.8, 128.8, 128.3, 126.4, 122.4, 114.3,
55.5, 40.6, 35.9, 32.1, 31.2, 30.9, 30.1, 29.7, 29.3, 20.2, 19.9, 17.9; HRMS (m/z): [M+H]+ calcd
for C32H43N5O4S: 593.3036; found: 593.3039.
2-((R)-3-((S)-2-(3-(4-Chlorophenyl)ureido)-N,3-dimethylbutanamido)-4-methylpentyl)-N-phe
nethylthiazole-4-carboxamide (8p).
Prepared from 7c by the same method as described in 8a. White solid, (0.06 g, 33%); mp
1
81.5-83.4 °C; H NMR (400 MHz, CDCl3): δ 8.03 (s, 1H, CH), 7.82 (s, 1H, NH), 7.38-7.35 (m,
1H, NH), 7.31-7.28 (m, 1H, ArH), 7.25 (d, J = 8.8 Hz, 2H, ArH), 7.23-7.16 (m, 4H, ArH), 7.11 (d,
J = 8.8 Hz, 2H, ArH), 6.47-6.36 (m, 1H, NH), 4.76-4.65 (m, 1H, CH), 3.69-3.60 (m, 1H, CH),
3.05 (s, 3H, NCH3), 2.94-2.93 (m, 1H, CH), 2.88-2.86 (m, 2H, CH2), 2.80-2.77 (m, 1H, CH),
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2.75-2.73 (m, 1H, CH), 2.14-2.11 (m, 1H, CH), 2.04-2.00 (m, 1H, CH), 1.83-1.81 (m, 1H, CH),
1.72-1.68 (m, 1H, CH), 1.24 (s, 1H, CH), 1.03 (d, J = 8.0 Hz, 3H, CH3), 1.0 (d, J = 8.0 Hz, 3H,
CH3), 0.98 (d, J = 6.6 Hz, 3H, CH3), 0.87 (d, J = 6.6 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3):
δ 175.3, 169.8, 169.8, 161.2, 161.1, 155.6, 149.6, 139.0, 137.9, 128.8, 128.6, 126.5, 122.5, 120.5,
55.6, 40.6, 35.9, 30.9, 30.1, 29.7, 29.2, 27.7, 20.2, 19.8, 18.0; HRMS (m/z): [M+H]+ calcd for
C31H40ClN5O3S: 597.2540; found: 597.2544.
2-((R)-3-((S)-2-(3-(4-Chloro-3-(trifluoromethyl)phenyl)ureido)-N,3-dimethylbutanamido)-4-m
ethylpentyl)-N-phenethylthiazole-4-carboxamide (8q).
Prepared from 7c by the same method as described in 8a. White solid, (0.05 g, 26%); mp
1
63.5-65.7 °C; H NMR (400 MHz, CDCl3): δ 8.03 (s, 1H, CH), 7.82 (s, 1H, NH), 7.73-7.70 (m,
1H, NH), 7.63-7.59 (m, 1H, NH), 7.52-7.45 (m, 1H, ArH), 7.40-7.34 (m, 1H, ArH), 7.30-7.27 (m,
3H, ArH), 7.23-7.17 (m, 3H, ArH), 4.76-4.66 (m, 1H, CH), 4.29 (t, J = 6.7 Hz, 1H, CH), 3.67 (d, J
= 6.9 Hz, 1H, CH), 3.02 (s, 3H, CH3), 2.91 (d, J = 7.1 Hz, 1H, CH), 2.82-2.78 (m, 1H, CH),
2.75-2.71 (m, 1H, CH), 2.37-2.34 (m, 1H, CH), 2.19-2.14 (m, 1H, CH), 2.08-2.02 (m, 1H, CH),
1.60 (s, 1H, CH), 1.24 (s, 1H, CH), 1.04 (d, J = 6.7 Hz, 3H, CH3), 0.98 (d, J = 6.4 Hz, 3H, CH3),
0.88 (d, J = 4.4 Hz, 3H, CH3), 0.85 (d, J = 6.8 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3):
δ176.6, 169.5, 168.6, 160.0, 159.4, 155.1, 154,6, 149.6, 139.8, 138.5, 131.2, 129.1, 128.6 126.9,
124.8 (CF3), 48.9, 35.6, 30.7, 29.7, 28.6, 24.8, 23.7, 21.2, 29.2, 18.8; HRMS (m/z): [M+H]+ calcd
for C32H39ClF3N5O3S: 665.2414; found: 665.2419.
4.2 Cell viability assay
The mitochondrial-dependent reduction of MTT to formazan was used to measure the
cytotoxicity. Briefly, cells were seeded in multiple 96-well plates at the density of 4 × 104 /mL.
After overnight of incubation, the cells were treated with serially diluted test compounds for 72 h.
After treatment, MTT solution (5 mg/mL) was added to each well, incubation was performed in
the dark, at 37°C for another 4 h. Supernatant was removed, and DMSO was then added to
dissolve the MTT formazan product. Its absorbance was assessed at 570 nm using a Molecular
Devices (USA) SpectraMax M5 spectrophotometer. Relative cell viability rates were calculated
versus untreated controls, with 50% inhibitory concentration (IC50) values calculated using Graph
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Pad Prism 5 (Graph Pad Software Inc., USA).18
4.3 Molecular docking
The crystal structure of microtubules and tubulysin M (PDB code: 4ZOL) was downloaded from
RCSB Protein Data Bank. Before the docking process, the natural co-crystalized ligand was
extracted and water molecules were removed from the crystal structure, H atoms were added and
side chains were fixed during protein preparation. All the steps parameters were set to the default
value of 100 in the window of staged minimization. Subsequently, the protein was prepared using
Biopolymer module implemented in Sybyl. Protein structure minimization was performed by
applying the Tripos force field and partial atomic charges were calculated by the Gasteiger-Huckel
method. The gradient of termination was defined as 0.005 kcal/(mol*A) and the maximum number
of interactions were 10000.
Conflict of interest
The authors declare have no conflict of interest.
Acknowledgment
The authors would like to thank the Doctoral Foundation of Yantai University (No. YX13B04),
and the Talent Development Project of Blue Economic Zone of Shandong Province (No.
RS11YX) for financial support.
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Table 1. Structures and antitumor activities of urea analogs
O
H
O
R3HN
N
N
N
O
R2
S
NHR1
IC50 (μM) (n =3, X±SD)
MCF-7
Compd. R1
R2
R3
NCl-H157
2.35±0.39
1.47±0.17
0.09±0.01
0.98±0.11
1.05±0.15
0.46±0.05
0.46±0.02
0.02±0.005
0.32±0.01
0.07±0.01
8a
8b
8c
8d
8e
8f
Me
CH2CH(CH3)2 p-OMeC6H4
CH2CH(CH3)2 p-ClC6H4
1.75±0.23
1.07±0.15
0.05±0.009
1.27±0.16
0.79±0.01
0.53±0.01
0.35±0.009
0.01±0.003
0.27±0.01
0.09±0.01
Me
Me
CH2CH(CH3)2 3-CF3-4-ClC6H3
CH2CH(CH3)2 p-OMeC6H4
CH2CH(CH3)2 p-ClC6H4
PhCH2
PhCH2
PhCH2
CH2CH(CH3)2 3-CF3-4-ClC6H3
8g
8h
8i
PhCH2CH2 CH2CH(CH3)2 p-NO2C6H4
PhCH2CH2 CH2CH(CH3)2 p-ClC6H4
PhCH2CH2 CH2CH(CH3)2 p-OMeC6H4
PhCH2CH2 CH2CH(CH3)2 3-CF3-4-ClC6H3
8j
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8k
8l
Me
CH3CHCH3
CH3CHCH3
CH3CHCH3
CH3CHCH3
p-OMeC6H4
p-ClC6H4
3-CF3-4-ClC6H3
p-OMeC6H4
p-OMeC6H4
p-ClC6H4
3-CF3-4-ClC6H3
/
2.31±0.57
1.59±0.19
0.76±00.6
1.02±0.15
0.97±0.11
0.38±0.02
0.11±0.03
0.001
4.31±0.87
2.34±0.31
0.62±0.15
2.05±0.37
0.69±0.01
0.12±0.005
0.09±0.01
0.001
Me
8m
8n
8o
Me
PhCH2
PhCH2CH2 CH3CHCH3
PhCH2CH2 CH3CHCH3
PhCH2CH2 CH3CHCH3
8p
8q
1
/
/
/
/
Taxol
/
0.01
0.01
Fig. 1: The design of pretubulysin analogs
Mep
Ile
Tuv
Tup
O
O
O
H
H
O
O
N
N
N
N
Ph
N
N
Ph
N
N
H
CH3 O
S
HN
O
CH3
S
HN
OEt
O
COOH
3
, IC50 = 0.47 M
1
, Pretubulysin
this work
O
H
N
H
N
F3C
Cl
O
N
N
H
R3HN
N
O
O
N
O
N
Ph
NHR1
O
R2 CH3
S
S
HN
2
I
, IC50 = 0.2 M
Urea analogues of Pretubulysin
Scheme 1. Synthesis of compounds 8a-8q.
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O
O
N
a, b
c
d,e, f
O
Br
Boc
Br
OEt
N
N
S
OEt
S
OEt
O
4
5
6
O
g, h
H
N
O
O
Boc
R3HN
N
N
N
N
S
O
R2
S
NHR1
NHR1
7
8a-8q
7a,
R1=CH3
7b
, R1=CH2Ph
7c
, R1=CH2CH2Ph
Scheme 1. Reagents and conditions: (a). thioacetamide, EtOH, r.t. 82%; (b). NBS, BPO, CCl4,
reflux, 64%; (c). 1). triphenylphosphine, Tol, reflux, 75%; 2). t-BuOK,
(S)-N-tert-butoxycarbonyl-N-methylvalinal, DCM, r.t. 69%; (d). H2, Pd/C, MeOH, r.t. 98%; (e).
LiOH, THF/H2O, r.t. 92%; (f). EDC, HOBt, R1NH2, r.t.74-85%; (g). TFA, DCM, r.t. 89%; (h).
EDC, HOBt, DCM, R3NHCONHCH(R2)CO2H, r.t. 63-75%.
Fig. 2. Predicted binding mode of pretubulysin (yellow, A), 8c (yellow, B), and 8h (yellow, C)
with tubulin. Dashed red lines indicate the H-bond interaction.
（A）
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（B）
(C)
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