Discovery of highly potent and selective small molecule ADAMTS-5 inhibitors that inhibit human cartilage degradation via encoded library technology (ELT)

Article

pubs.acs.org/jmc

Discovery of Highly Potent and Selective Small Molecule ADAMTS‑5

Inhibitors That Inhibit Human Cartilage Degradation via Encoded

Library Technology (ELT)

Hongfeng Deng,*^,†Heather O’Keefe,^†Christopher P. Davie,^†Kenneth E. Lind,^†Raksha A. Acharya,^†,∞

G. Joseph Franklin,^†Jonathan Larkin,^‡Rosalie Matico,^§Michael Neeb,^∥Monique M. Thompson,^§

Thomas Lohr,^‡Jeﬀrey W. Gross,^⊥Paolo A. Centrella,^†,×Gary K. O’Donovan,^†Katie L. (Sargent) Bedard,^†

Kurt van Vloten,^†,●Sibongile Mataruse,^†Steven R. Skinner,^†Svetlana L. Belyanskaya,^†

Tiﬀany Y. Carpenter,^#Todd W. Shearer,^#Matthew A. Clark,^†,×John W. Cuozzo,^†,×

Christopher C. Arico-Muendel,*^,†and Barry A. Morgan^†

^†ELT Boston, Platform Technology and Science, GlaxoSmithKline, Waltham, Massachusetts, United States

^‡Biopharm Discovery, Medicines Discovery and Development, GlaxoSmithKline, Upper Merion, Pennsylvania, United States

^§Biological Reagent and Assay Development, Platform Technology and Science, GlaxoSmithKline, Upper Providence, Pennsylvania,

United States

^∥Center of Excellence for External Drug Discovery, Medicines Discovery and Development, GlaxoSmithKline, Upper Merion,

Pennsylvania, United States

^⊥Screening and Compound Proﬁling, Platform Technology and Science, GlaxoSmithKline, Upper Providence, Pennsylvania, United

States

^#Muscle Metabolism Discovery Performance Unit, Medicines Discovery and Development, GlaxoSmithKline, Research Triangle Park,

North Carolina, United States

S

* Supporting Information

ABSTRACT: The metalloprotease ADAMTS-5 is considered a potential target for the treatment of osteoarthritis. To identify

selective inhibitors of ADAMTS-5, we employed encoded library technology (ELT), which enables aﬃnity selection of small

molecule binders from complex mixtures by DNA tagging. Selection of ADAMTS-5 against a four-billion member ELT library

led to a novel inhibitor scaﬀold not containing a classical zinc-binding functionality. One exemplar, (R)-N-((1-(4-(but-3-en-1-

ylamino)-6-(((2-(thiophen-2-yl)thiazol-4-yl)methyl)amino)-1,3,5-triazin-2-yl)pyrrolidin-2-yl)methyl)-4-propylbenzenesulfona-

mide (8), inhibited ADAMTS-5 with IC₅₀= 30 nM, showing >50-fold selectivity against ADAMTS-4 and >1000-fold selectivity

against ADAMTS-1, ADAMTS-13, MMP-13, and TACE. Extensive SAR studies showed that potency and physicochemical

properties of the scaﬀold could be further improved. Furthermore, in a human osteoarthritis cartilage explant study, compounds

8 and 15f inhibited aggrecanase-mediated ³⁷⁴ARGS neoepitope release from aggrecan and glycosaminoglycan in response to IL-

1β/OSM stimulation. This study provides the ﬁrst small molecule evidence for the critical role of ADAMTS-5 in human cartilage

degradation.

agents that can modify the progression of the disease.¹OA is

INTRODUCTION

■

characterized by the degradation of articular cartilage as a result

Human osteoarthritis (OA) is a degenerative joint disease

aﬀecting tens of millions of people worldwide. Current

of uncontrolled proteolytic destruction of extracellular matrix.²

treatments are mostly conﬁned to reducing inﬂammation and

pain associated with the disease using steroidal and non-

steroidal anti-inﬂammatory drugs (NSAIDs) with few available

Received: March 30, 2012

Published: August 14, 2012

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Scheme 1. Synthesis of the Four Cycle DNA Encoded Triazine Library

The major components of the cartilage extracellular matrix are

type II collagen and aggrecan.³Aggrecan is a large multidomain

proteoglycan that provides cartilage with elasticity and

compressibility; thus, its loss from the matrix is considered a

critical event in the early stages of cartilage destruction.⁴

Aggrecanases including ADAMTS-4 (aggrecanase-1) and

ADAMTS-5 (aggrecanase-2) are members of the ADAMTS

(a disintegrin and metalloproteinase with thrombospondin

motifs) family of zinc metalloproteases that cleave the N-

terminal interglobular domain of aggrecan at the Glu³⁷³-Ala³⁷⁴

peptide bond.^3,5,6Inhibition of aggrecanases reduces aggrecan

degradation and leads to overall cartilage protection.^7,8

However, the identity of the aggrecanase isozyme playing the

dominant role in cartilage destruction remains controversial.

On the one hand, ADAMTS-5 knockout mice and ADAMTS-

4/-5 double knockout mice, but not ADAMTS-4 knockout

mice, were shown to be signiﬁcantly protected from

degradation,⁹⁻¹¹suggesting that ADAMTS-5 is the crucial

enzyme in aggrecanolysis. On the other hand, small interfering

RNA (siRNA) speciﬁcally targeting either ADAMTS-4 or

ADAMTS-5 attenuated the degradation of aggrecan in both

cytokine-stimulated normal cartilage and unstimulated human

OA cartilage, suggesting that both aggrecanases participate in

cartilage degradation.¹²To further elucidate these enzymes’

respective roles in cartilage degradation and validate them as

targets for OA prevention and treatment, we launched a

campaign to identify speciﬁc small molecule inhibitors for

either ADAMTS-4 or ADAMTS-5 using our proprietary

encoded library technology (ELT). Most of the known

aggrecanase inhibitors contain known zinc binding groups

(carboxylic acids, hydroxamic acids, tartrates, hydantoins, etc.)

and possess limited selectivity over other zinc metalloproteases

as well as poor physicochemical properties for systemic

exposure.^1,13In this paper we report the discovery of potent

and selective novel ADAMTS-5 inhibitor scaﬀold lacking a

classical zinc binding motif that inhibits human cartilage

degradation in an ex vivo human OA cartilage explant

experiment.

RESULTS AND DISCUSSION

■

ELT is a novel hit identiﬁcation technology that has recently

attracted signiﬁcant attention from both academia and

industry.¹⁴⁻¹⁶The technology encompasses four critical

components: (1) the design and synthesis of DNA encoded

small molecule libraries as combinatorial mixtures of 10⁶−10¹⁰

members, (2) aﬃnity selection of libraries against therapeutic

targets, (3) hit structure deconvolution through DNA

sequencing, and (4) oﬀ-DNA hit resynthesis and activity

conﬁrmation. The advantages of this approach include high

diversity, minimal target protein consumption, and rapid

execution; following library synthesis (on a scale amenable to

several thousand target screens), the complete process of

aﬃnity selection, deconvolution, and hit conﬁrmation may be

accomplished in 6 weeks. Since its inception, ELT has identiﬁed

tractable hit series for several dozen targets including both

soluble and integral membrane proteins.¹⁷⁻²⁰Application of

ELT to ADAMTS-4 has been previously reported.^20,21Here we

describe the identiﬁcation of ADAMTS-5 inhibitors from a

four-billion-member DNA encoded triazine library.

The library scaﬀold for the present ADAMTS-5 work

consists of a 1,3,5-triazine core elaborated with four synthetic

cycles to create diversity. As we previously reported for two

related libraries,¹⁷it was synthesized using the split-and-pool

strategy, beginning from a short sequence of duplex DNA

stabilized by a synthetic hairpin (the “headpiece”). The design

of the headpiece allows buildup of a small molecule warhead

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Figure 1. Spotﬁre cube data view of ADAMTS-5 selection features: BB1, cycle 1 building blocks; BB2, cycle 2 building blocks; BB4, cycle 4 building

blocks; BB3, cycle 3 building blocks. The BB3 building blocks are represented by diﬀerent colors.

starting from the free amino group, while DNA tags encoding

the building blocks (BBs) are enzymatically ligated at the

opposite end. In this instance, cycle 1 incorporated 192 Fmoc-

amino acids via acylation onto the AOP-headpiece (DNA)

starting material (1, Scheme 1; refer to Supporting Information

Figure 2 for detailed AOP-headpiece structure). After Fmoc-

deprotection (2), the triazine scaﬀold was installed by the

addition of cyanuric chloride. In cycle 2, a set of 479 amine

building blocks were incorporated (3), followed by a set of 96

diamines (including 60 o-nitroveratryloxycarbonyl (Nvoc)

diamines) at cycle 3 (4). After removal of the photolabile

Nvoc protecting group, the free amines were then further

elaborated in cycle 4 by acylation with 173 carboxylic acids,

reductive alkylation with 94 aldehydes, sulfonylation with 107

sulfonyl chlorides, and urealaytion with 85 isocyanates (5). The

DNA sequence was then “closed” with a ﬁnal tag encoding a

library identiﬁer, a priming region for PCR ampliﬁcation, and a

degenerate sequence to control for PCR ampliﬁcation biases.

The aﬃnity selections were performed on a chemically

biotinylated human ADAMTS-5(262−624). The target protein

construct (∼10 μg total) was immobilized on Phynexus aﬃnity

columns derivatized with streptavidin. An aliquot (∼5 nmol) of

library was incubated on the tips for an hour, and the tips were

then washed extensively to remove nonbinders. The bound

molecules were eluted by heat denaturation of the protein at 80

°C. The eluant was then incubated with fresh immobilized

protein to start the second round of aﬃnity selection. Three

rounds of selection were performed. In order to exclude

molecules whose enrichment was due to binding to the resin

matrix or streptavidin, a parallel selection performed in an

identical fashion without the addition of the protein target was

used as a no target control (NTC). In order to add priming

sites for DNA sequencing and to ensure enough molecules for

sequencing, the ﬁnal eluant was ampliﬁed by PCR from 3 × 10⁵

to 1 × 10⁷molecules. DNA sequencing was then performed

using Roche/454 technology, and the sequences were

translated to identify the structures of the putative ADAMTS-

5 binders.

Further analysis of the aﬃnity selection output could be

conveniently performed in a Spotﬁre cube view. In Figure 1,

warheads enriched at the level of two independent DNA

sequences or greater are shown with the cube axes representing

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a

Scheme 2. Synthesis of ADAMTS-5 Oﬀ-DNA Hits

a

Reagents and conditions: (i) sulfonyl chlorides (R₄SO₂Cl), triethylamine, dichloromethane; (ii) 20% triﬂuoroacetic acid in dichloromethane; (iii)

(a) cycle 2 amines (NR₂, 1.0 equiv), DIPEA (1.0 equiv), acetonitrile, 0 °C, 30 min; (b) cycle 3 amines (NR₃, 1.0 equiv), DIPEA (3.0 equiv),

acetonitrile, room temperature, 1 h; (c) cycle 1 amines (NR₁, 3 equiv), DIPEA (3 equiv), acetonitrile, 80 °C, 3 h to overnight.

a

Scheme 3. Alternative Synthesis of ADAMTS-5 Oﬀ-DNA Hits

a

Reagents and conditions: (i) (a) cycle 2 amines (NR₂, 1.0 equiv), DIPEA (1.0 equiv), acetonitrile, 0 °C, 30 min; (b) (R)-tert-butyl (pyrrolidin-2-

ylmethyl)carbamate (1.0 equiv), DIPEA (3.0 equiv), acetonitrile, room temperature, 1 h; (c) cycle 1 amines (NR₁, 3 equiv), DIPEA (3.0 equiv),

acetonitrile, 80 °C, 3 h to overnight; (ii) 20% triﬂuoroacetic acid in dichloromethane; (iii) sulfonyl chlorides (R₄SO₂Cl), triethylamine,

dichloromethane.

building blocks used in cycle 1, cycle 2, and cycle 4 chemistry,

respectively, and with cycle 3 building blocks indicated by

diﬀerent colors. As discussed in the earlier report,¹⁷families of

related warheads are of particular interest because they contain

one or more building blocks in common and are easily

recognized in these views as lines or planes. Here, two such

features were evident: the bright green line corresponding to

scaﬀold 6a and the dark green line corresponding to scaﬀold

6b. A detailed examination of the data set revealed that all of

the putative binders on the 6a line had the same BB1 (2-

aminopent-4-enoic acid) and BB2 ([2-(thiophen-2-yl)thiazol-4-

yl]methanamine) moieties. In addition, all of the binders on the

6a line also shared a common BB at cycle 3, ((R)-pyrrolidin-2-

ylmethanamine). These families varied in the nature of the BB4

building blocks. A closer check of these BB4s, however, found

that all 23 groups selected at this site were sulfonyl chlorides,

whereas sulfonyl chlorides comprised 107 of the 459 building

blocks available. Therefore, the selection for this site was also

speciﬁc. The dark green line (line 6b) in this cube view was

analyzed in the same fashion, showing that 2-amino-3-

(naphthalen-2-yl)propanoic acid, (S)-3-aminoazepan-2-one,

and (R)-pyrrolidin-3-amine were selected at cycles 1, 2, and

3, respectively.

medicinal chemistry methods. Compounds were prepared using

two similar synthetic routes as outlined in Schemes 2 and 3. In

Scheme 2, the N¹-Boc protected 2-(aminomethyl)pyrrolidine

(I) was reacted with various sulfonyl chlorides followed by

removal of the Boc group to give corresponding amines (III,

NR₃). Three sequential amine replacements of cyanuric

chloride under diﬀerent conditions aﬀorded ﬁnal compounds

IV in reasonable yields (60−80%). Alternatively, as in Scheme

3, the cyanuric chloride was sequentially reacted with three

diﬀerent amines under diﬀerent conditions to give intermediate

compounds V, which were then deprotected and reacted with

various sulfonyl chlorides to yield ﬁnal compounds IV in 25−

65% overall yield.

Next, we tested these oﬀ-DNA compounds for their ability to

inhibit ADAMTS-5 in a ﬂuorescence resonance energy transfer

(FRET) assay using human ADAMTS-5 protein and WAAG-

3R substrate.²²As shown in Figure 2, three compounds from

the 6a series including 7, 8, and 9, although having diﬀerent

cycle 4 groups, all showed potent inhibitory activity against

ADAMTS-5 with IC₅₀values of 50, 30, and 40 nM, respectively.

Oﬀ-DNA compounds 10 and 11 from the 6b series were

inactive (both having IC₅₀> 10 uM), suggesting that these

binders might bind to the protein at a site that does not aﬀect

the protein’s enzymatic activity (since ELT selections are based

on binding aﬃnity between library compounds and a protein

target without a functional readout, it is possible to ﬁnd good

binders with no functional activity). This result was very

encouraging, especially considering that among more than four-

The above analysis suggested that at least two series of

compounds were selected from the library by ADAMTS-5

through aﬃnity interaction. To conﬁrm this analysis, we

synthesized representative compounds from each of the two

diﬀerent series without the DNA attachment by standard

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Figure 2. Structures of initially synthesized oﬀ-DNA compounds and the corresponding activity against ADAMTS-5. Superscript “a” indiates that all

IC₅₀values are the mean of at least three independent experiments. IC₅₀> 10 μM for compounds 10 and 11.

billion diversiﬁed compounds in the library, only a small

number of compounds were selected based on speciﬁc binding

interactions with ADAMTS-5. In addition, this novel and

potent ADAMTS-5 inhibitor scaﬀold contains no classical zinc

binding groups typically seen in known metalloprotease

inhibitors and is therefore an attractive starting point for

achieving high speciﬁcity and druglike properties.^23,24

The aﬃnity selection data aﬀord an initial insight into

structure−activity relationships (SARs) for this novel

ADAMTS-5 inhibitor scaﬀold. To extend our understanding

beyond the building blocks contained within the library and

also to deconvolute any biases that could be introduced by the

DNA linkage of the warheads, we undertook further SAR

studies beginning with each of the attached building blocks in

turn. In the absence of X-ray cocrystal structures, this eﬀort was

carried out in traditional medicinal chemistry fashion.

butylamine, 1-cyclopropylmethanamine, methylamine, hydrox-

yl, or hydrogen (R₁in 12) to map the scope of tolerance to

structural changes at this site. From Table 1 we can see that

replacing the 3-butene-1-amine group with n-butylamine (12a)

or 1-cyclopropylmethanamine (12b) decreased the activity by

5- and 2-fold correspondingly. Truncation of this side chain to

methylamine (12c), hydroxyl group (12d), and hydrogen

(12e) also decreased the activity signiﬁcantly, suggesting the

necessity of a small alkyl or alkenyl amine substitution at this

site. This initial observation was conﬁrmed with additional SAR

studies as shown in the second part of Table 1, using 13 as a

generic structure where a n-propylamine (13a) or a cyclopropyl

substitution (13e) retained the activity and an isobutylamine

substitution (13c) gave similar activity as the 1-cyclo-

propylmethanamine substitution (12b). We note that at the

triazine 4-position, the 2-thiophenythiazole group and the 2-

phenylthiazole group gave equal potency as shown in Table 2,

compounds 7 and 14a. Further increase of the ring size to a

cyclopentyl group (13f) decreased the activity by 30-fold, and

The SAR around the triazine 2-position (BB1 site) was

explored ﬁrst. As can be seen from Table 1, the but-3-en-1-

amine group in compound 7 was initially replaced by n-

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a

Table 1. SAR for Triazine 2-Position (BB1 Site)

a

All IC₅₀values are the mean of at least three independent experiments.

changing the primary amine to a secondary amine (13b)

completely abolished the activity. While certain small sized

aminoalkyl or aminoalkenyl substitutions were preferred, an

appropriate heteratom/hydrophilic group at the distal end of a

substituent was acceptable. For example, the 3-aminopropane-

nitrile substitution (13g) at this site gave potency comparable

to that of compound 7. But the hydroxyl (13h) and methoxy

groups (13i) both decreased the potency by 5-fold, and the

(tetrahydro-2H-pyran-4-yl)methyl substitution (13j) rendered

the compound 40-fold less active.

thiazole ring with a second phenyl ring, such as appeared in 4-

or 3-biphenylmethyl groups (14b and 14c) rendered the

compounds totally inactive. Removal of the distal thiophene

ring decreased the activity (14d) about 8-fold. Replacement of

the 2-methylthiazolemethyl with a simple benzyl group gave

further decreased activity (14e). These observations led us to

consider whether it was the thiazole ring at this position or

rather the basic nitrogen in the thiazole ring that was crucial for

the activity. This issue was further explored with additional

single ring systems as shown in Table 2, using 15 as generic

structure. Using compound 12a as a reference, we found that

simple thiophene (15a) and substituted thiophenes (15b, 15c)

were much less active (or inactive) than the original thiazole

derivatives. On the other hand, the 2-(6-methyl)pyridyl group

We then explored the triazine 4-position (BB2) as shown in

Table 2. As can be seen, the 2-thiophenylthiazolemethyl group

can be replaced by the phenylthiazolemethyl group with good

retention of activity (14a). However, further replacement of the

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a

Table 2. SAR for Triazine 4-Position (BB2 Site)

a

All IC₅₀values are the mean of at least three independent experiments.

(15d) gave potency comparable to that of the 2-thiophenylth-

iazole biaryl ring system at this position, which suggests that the

nitrogen in the two ring system could be the key to the

observed activity. This notion was further supported by the

comparison between compounds 15d and 15e, where the two

ring systems should have the same steric eﬀect and the missing

nitrogen in 15e rendered the compound inactive. More

importantly, the potency was signiﬁcantly boosted (more

than 8-fold) when the (1-methyl-1H-imidazole)-4-methyl

group (15f), a small but nitrogen containing aryl ring, was

adopted at this position. An additional beneﬁt of this

modiﬁcation is that it signiﬁcantly reduced the compound’s

molecular weight and cLogP (by ∼80 and 2.5, respectively).

Being able to improve physicochemical properties while

maintaining a compound’s potency opened the window for

productive lead optimization. The last three compounds in this

table with an alkyl or cycloalkyl group (15h, 15i, and 15j) at

the triazine 4-position proved to be much less active.

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a

Table 3. SAR for Triazine 6-Position (BB3 Site)

a

All IC₅₀values are the mean of at least three independent experiments.

The structural exploration around triazine 6-position (BB3

site) was summarized in Table 3. At this position, we

investigated the pyrrolidine ring for its substitution position

and stereochemistry. As can be seen, switching the active

conﬁguration of the (R)-pyrrolidin-2-ylmethanamine group (7)

to its enantiomer (S)-pyrrolidin-2-ylmethanamine group (16)

caused a 30-fold drop in potency. Meanwhile, moving the

substitution from the pyrrolidine 2-postion to 3-position (17

and 18) led to a complete loss of activity, suggesting that the

(R)-pyrrolidin-2-ylmethanamine group at this position is highly

speciﬁc.

Lastly, we explored SAR around the sulfonamide moiety. An

examination of the initial three oﬀ-DNA compounds 7, 8, and 9

(Figure 2) suggests that this site tolerated some variations, with

both β-naphthalenyl and 4-triﬂuoromethyl or 4-propyl

substituted phenylsulfonamides giving almost equal potency.

Because of the simplicity of the phenyl ring system, our SAR

study focused on investigating diﬀerent substitution eﬀects on

the phenyl ring as shown in Table 4. When a phenyl group was

adopted, p-CF₃and p-OCH₃substitutions (14a and 19c) gave

4-fold increase in potency compared to no substitution (19d).

Both m- and o-CF₃substitutions yielded inactive compounds

(19a, 19b). Further studies varied substituent sizes, electronic

nature, and hydrogen-bonding capacities as represented by

generic structure 20 in Table 4. As can be seen, chloro

substitutions gave potency comparable to that of compound

12a, with the para-position (20a) being slightly better than the

meta-position (20b). A similar activity was seen for 3,4-

dimethyl substitution (20c). A second phenyl group sub-

stitution at the para-position (20d) decreased the activity

further, as did the ﬂuoro (20h) and some hydrophilic

substitutions including MeSO₂, CN, and OH (20e, 20f, 20g,

20i, and 20j). Overall, an appropriate lipophilic group at the

phenyl para-position favored activity. When the phenyl ring was

replaced by a cyclopropyl group (19e), the compound became

inactive.

As demonstrated above, the scaﬀold has deﬁned SAR and

tolerates speciﬁc structural variations. We next proﬁled these

compounds for ADAMTS-5 selectivity over other related zinc

metalloproteinases as illustrated in Table 5. All of the tested

compounds were more than 10-fold selective for ADAMTS-5

over ADAMTS-4 and more than 88-fold selective for

ADAMTS-5 over other related enzymes including TACE,

ADAMTS-1, and MMP-13. The selectivity demonstrated by

these novel ADAMTS-5 inhibitors is very important, since, so

far, few selective ADAMTS-5 small molecular inhibitors have

been reported, especially relative to ADAMTS-4 because of the

high sequence homology and conformational similarity of their

active sites revealed by X-ray crystallography.²⁵This, together

with the unprecedented scaﬀold structure, renders the

inhibitory mechanism of these molecules of considerable

interest. The inhibitory potency of two exemplars, 8 and 14a

was essentially unaﬀected by added zinc over the range 1−100

μM (Supporting Information), in agreement with the lack of

obvious zinc binding moiety. In addition, an enzyme kinetic

study with compound 8 revealed that it inhibits ADAMTS-5 in

a peptide substrate competitive manner (Supporting Informa-

tion). Further discernment of the mechanism of action will be

enabled by crystallographic and biophysical approaches.

Meanwhile, the identiﬁcation of these potent and selective

ADAMTS-5 inhibitors in a very short period of time

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a

Table 4. SAR for Sulfonamide Moiety (BB4 Site)

a

All IC₅₀values are the mean of at least three independent experiments.

demonstrated ELT’s utility as a powerful technology for rapid

and eﬃcient hit/lead identiﬁcation.

In vitro proﬁling of three representative compounds 15f, 13g,

and 13e showed low solubility (16, 6, and 2 μM, respectively),

high to low permeability (3.5 × 10⁻⁵, <5 × 10⁻⁶, < 5 × 10⁻⁶

cm/s, respectively) and low microsomal stability (Cl_int,apparentof

2930, 1799, and 1596 mL min⁻¹kg⁻¹, respectively, rat

microsomes).²⁶An in vivo rat pharmacokinetic (PK) study

was also performed. As shown in Table 6, these compounds

demonstrated a range of C_max, AUC, clearance, Vd_ss, T_1/2, and

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a

Table 5. Selectivity Data for Selected Compounds

IC₅₀, μM (fold of selectivity)

IC₅₀, μM

ADAMTS-5

compd

ADAMTS-4

TACE

ADAMTS-1

MMP-13

7

0.050

0.030

0.040

0.070

0.090

0.030

1.87 (37)

1.50 (50)

6.0 (150)

8.3 (118)

1.2 (13)

1.3 (43)

>50 (>1000)

12.6 (180)

19.9 (221)

7.9 (263)

>50 (>1000)

>25 (>833)

>25 (>625)

7.9 (113)

8

9

13e

13g

15f

7.9 (88)

25 (833)

a

All IC₅₀values are the mean of at least three independent experiments.

a

Table 6. Rat PK Data Summary for Selected Compounds

compd

dose, iv/po (mg/kg)

C_max, iv (ng/mL)

AUC_0−t, iv (ng·h/mL)

Cl, iv (mL h⁻¹kg⁻¹

)

Vd_ss, iv (L/kg)

T_1/2, iv (h)

F (%)

15f

13g

13e

0.9/2.0

0.8/0.5

0.9/0.6

207

644

144

422

6537

1998

643

2.4

1.4

2.0

0.6

2.0

7.9

1.5

8.4

8.0

1797

1357

a

Rat cassette dosing, and all values are averages of three rats. F% indicates oral bioavailability.

Figure 3. ADAMTS-5 inhibitors suppress cytokine mediated cartilage degradation in human osteoarthritis cartilage explants. Time course of

³⁷⁴ARGS aggrecan neoepitope release (A) and GAG release (C) from cartilage disks treated with increasing concentrations of IL-1β and OSM is

suppressed in response to inhibitor compound treatment compared to DMSO control. Mean ³⁷⁴ARGS neoepitope (B) and GAG (D) release levels

are suppressed by inhibitor compound treatment compared to DMSO control across the entire 18 days of IL-1β and OSM stimulation.

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oral bioavailability despite sharing a common core structure,

triazine pyrrolidine(4-n-propanephenyl)sulfonamide. Com-

pound 13g showed high clearance (1998 mL h⁻¹kg⁻¹) and a

short half-life (2 h), while compound 13e displayed a moderate

clearance (643 mL h⁻¹kg⁻¹) and a signiﬁcantly increased half-

life (7.9 h). The only structural diﬀerence between the two

compounds is that the 2-cyanoethylamine group in 13g is

changed to the 2-cyclopropylamine group in 13e. The potential

improvement in PK properties suggests that these properties

were more related to diﬀerent side chains than to the central

triazine scaﬀold.²⁷

The high potency and selectivity make these inhibitors

attractive pharmacological tools for investigating ADAMTS-5

protein’s role in OA disease progression and treatment.

Accordingly, two of the above ADAMTS-5 inhibitors were

further evaluated for inhibition of cartilage degradation in an ex

vivo human OA cartilage explant system.^28,29Total knee

replacement surgical discard tissue from an OA patient was

obtained within 24 h following surgery, and cartilage was

processed from the bone, cut into uniform disks, randomized,

and placed into culture medium in 96-well plates. Following

equilibration with tissue culture medium, cartilage disks were

treated with ADAMTS-5 inhibitors 8 and 15f and, for

comparison, the published MMP/aggrecanase hydroxamate

inhibitor (2R,3R)-1-((4-((2-chloro-4-ﬂuorobenzyl)oxy)-

phenyl)sulfonyl)-N,3-dihydroxy-3-methylpiperidine-2-carboxa-

mide (21, Figure 4).²⁴All treatments and dose levels were

apparent decreased sensitivity of the GAG release end point

compared to the ³⁷⁴ARGS neoepitope has been consistently

observed in our experience using this system and has been

independently reported elsewhere³¹potentially owing to the

fact that GAG release measurements cannot diﬀerentiate

between GAG derived from synthetic or degradative sources.

Metabolic viability of the cartilage disks at the end of the

experiment, as measured by AlamarBlue ﬂuorescent conversion

assay, was unaﬀected by any of the treatments (data not

shown), suggesting minimal toxicity.

The above pharmacological evaluation of the representative

ADAMTS-5 inhibitors in the human OA cartilage explant assay

showed signiﬁcant inhibition of cartilage degradation as

evidenced by suppressed release of ³⁷⁴ARGS neoepitope and

GAG as a consequence of aggrecan degradation triggered by IL-

1β/OSM. With the caveat that the OA cartilage used here is

derived from a single patient and thus imperfectly represents

OA pathology, this experiment nonetheless represents, to our

knowledge, the ﬁrst demonstration of eﬃcacy in a disease

related model by small molecules with a high speciﬁcity for

ADAMTS-5. These data further support the role of ADAMTS-

5 as an important target for the treatment of human OA.

CONCLUSIONS

■

By use of our proprietary encoded library technology, a series

of potent and selective ADAMTS-5 inhibitors were rapidly

identiﬁed from a newly developed, DNA tagged, novel four-

billion member small molecule compound library through

aﬃnity-based selection. These inhibitors contain neither

carboxylate nor hydroxamate functional groups typically seen

in zinc metalloproteinase inhibitor scaﬀolds. Extensive SAR

study revealed the core structural requirements necessary for

ADAMTS-5 inhibition but also demonstrated good potential

for improving potency and physicochemical properties through

further structural optimization. More importantly, these

inhibitors were highly selective against other related enzymes

and were capable of inhibiting cartilage degradation in an

explant experiment, thus providing further support for the

importance of ADAMTS-5 as a therapeutic target for OA

prevention and treatment.

Figure 4. Structure of 21.

conducted in replicates of seven to control for inherent

cartilage plug variability. All samples were treated with

increasing levels of recombinant human IL-1β (interleukin-

1β)/OSM (oncostatin M, an interleukin-6 family cytokine) to

stimulate cartilage degradation, and conditioned culture

medium was removed at various time points and tested for

cartilage degradation markers.

As shown in Figure 3, ADAMTS-5 inhibitors 8 and 15f

demonstrated a dose dependent suppression of aggrecanase

speciﬁc ³⁷⁴ARGS neoepitope release compared to DMSO

control treatment over the course of the experiment (Figure 3A

and Figure 3B). The two inhibitors also showed suppression of

glycosaminoglycan (GAG) release over the DMSO control,

although the eﬀect was less signiﬁcant (Figure 3C and Figure

3D). In both cases, the nonselective metalloprotease inhibitor

21, which has an IC₅₀of 6.3 nM against ADAMTS-5, 3.0 nM

against ADAMTS-4, 0.9 nM against MMP-13, and 1.6 nM

against TACE, demonstrated the most potent inhibition of

cartilage degradation.³⁰The higher eﬃcacy achieved by 21 in

this experiment may be due to more eﬀective inhibition of

ADAMTS-5; alternatively, this compound may act in a

nonspeciﬁc manner against other aggrecan-cleaving proteases

that may have been stimulated under these conditions. The

EXPERIMENTAL SECTION

■

All solvents and reagents were used as obtained. ¹H NMR spectra were

recorded on a Varian NMR 300 M or a Varian Mercury 400 Plus.

Chemical shifts are expressed in parts per million (ppm, δ units).

Coupling constants (J) are in units of hertz (Hz). Splitting patterns

describe apparent multiplicities and are designated as s (singlet), d

(doublet), t (triplet), q (quartet), dd (double doublet), dt (double

triplet), m (multiplet), br (broad). Analytical purity was ≥95% unless

stated otherwise. The purity of ﬁnal compounds was checked using an

Agilent 1100 HPLC system coupled with a Thermo Finnigan LCQ

mass spectrometer. All mass spectra were performed by electrospray

ionization (ESI). Four diﬀerent HPLC conditions were used to analyze

compound purity. Method A involved a Phenomenex Luna 3 μm

C8(2), 30 mm × 3.00 mm 100A column, running gradient of 30−98%

MeCN/H₂O (+0.1% TFA) over 3.5 min with ﬂow rate of 0.7 mL/min.

Method B involved a Phenomenex Luna 3 μm C8(2), 100 mm × 3.00

mm 100A column, running gradient of 30−85% MeCN/H₂O (+0.1%

TFA) over 12 min with ﬂow rate of 0.7 mL/min. Method C involved a

Phenomenex Luna 3 μm C8(2), 100 mm × 3.00 mm 100A column,

running gradient of 10−85% MeCN/H₂O (+0.1% TFA) over 12 min

with ﬂow rate of 0.7 mL/min. Method D involved a SUNFIRE-C19,

50 mm × 2.1 mm, 3.5 μm column, running gradient of 5−100%

MeCN/H₂O (+0.01% TFA) over 1.5 min with ﬂow rate of 2 mL/min.

High-resolution mass measurement was performed on Bruker

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MicroTOF electrospray mass spectrometer coupled with an Agilent

1100 HPLC system. Puriﬁcation of ﬁnal compounds for biological

testing was performed on a Gilson GX-281 system with either a

Phenomenex Luna 5 μm C8(2), 100 mm × 21.20 mm 100A column,

running gradient of 10−95% MeCN/H₂O (+0.1% TFA) over 15−20

min with ﬂow rate of 22 mL/min (method E) or a Shimadzu, PRC-

ODS, 250 mm × 21.2 mm column, running gradient 20−70% MeCN/

H₂O (+0.05% TFA) over 8 min with ﬂow rate of 30 mL/min (method

F).

general method A. MS (ESI) m/z [M + H]⁺= 625.3, t_R= 7.75 min

(HPLC method B). ¹H NMR (400 MHz, CD₃OD) δ 7.69 (d, J = 8.4

Hz, 1H), 7.62−7.55 (m, 3H), 7.37 (s, 1H), 7.33 (d, J = 8.4 Hz, 1H),

7.25 (d, J = 8.0 Hz, 1H), 7.16−7.11 (m, 1H), 5.80 (m, 1H), 5.13−5.02

(m, 2H), 4.68 (m, 2H), 4.25 (m, 1H), 3.63 (m, 1H), 3.48 (m, 3H),

3.19−3.01 (m, 2H), 2.61 (m, 2H), 2.37 (m, 1H), 2.30 (m, 1H), 2.04

(m, 3H), 1.91 (m, 1H), 1.61 (m, 2H), 0.91 (dt, J = 7.6, 3.6 Hz, 3H).

HRMS (M + H)⁺calcd for [C₂₉H₃₆N₈O₂S₃+ H] 625.2196; found

625.2152

Synthesis of DNA Encoded Library. See Supporting Information

for a detail description including headpiece DNA structure, DNA tag

information, four sequential chemical conversions, and ﬁnal library

closing DNA sequence ligation.

N-({(2R)-1-[4-(3-Buten-1-ylamino)-6-({[2-(2-thienyl)-1,3-thia-

zol-4-yl]methyl}amino)-1,3,5-triazin-2-yl]-2-pyrrolidinyl}-

methyl)-2-naphthalenesulfonamide (9). By use of {[2-(2-

thienyl)-1,3-thiazol-4-yl]methyl}amine as NR₂, N-[(2R)-2-pyrrolidi-

nylmethyl]-2-naphthalenesulfonamide TFA salt as NR₃, and 3-buten-

1-amine as NR₁, the title compound was prepared by following general

method A. MS (ESI) m/z [M + H]⁺= 633.2, t_R= 6.97 min (HPLC

method B). HRMS (M + H)⁺calcd for [C₃₀H₃₂N₈O₂S₃+ H]

633.1883; found 633.1853.

3-Fluoro-4-methyl-N-((R)-1-(4-((2-(naphthalen-1-yl)ethyl)-

amino)-6-(((S)-2-oxoazepan-3-yl)amino)-1,3,5-triazin-2-yl)-

pyrrolidin-3-yl)benzenesulfonamide (10). By use of (S)-3-amino-

azepan-2-one as NR₂, (R)-3-ﬂuoro-4-methyl-N-(pyrrolidin-3-yl)-

benzenesulfonamide TFA salt as NR₃, and 2-(naphthalen-1-

yl)ethanamine as NR₁, the title compound was prepared by following

general method A. MS (ESI) m/z [M + H]⁺= 633.2, t_R= 2.31 min

(HPLC method A). ¹H NMR (400 MHz, DMSO-d₆) δ 8.13 (m, 1H),

8.07 (m, 1H), 7.86 (m, 3H), 7.71 9 (m, 1H), 7.56 (m, 3H), 7.47 (m,

2H), 7.41 (m, 1H), 4.47 (m, 1H), 3.84 (m, 1H), 3.75−3.35 (m, 9H),

3.13 (m, 1H), 3.01 (m, 2H), 2.31 (s, 3H), 2.10−1.60 (m, 6H), 1.39

(m, 1H), 1.24 (m, 1H). HRMS (M + H)⁺calcd for [C₃₂H₃₈N₈O₃FS +

H] 633.2772; found 633.2773.

Synthesis of Compounds oﬀ-DNA. General Method A. To a

solution of a desired sulfonyl chloride (1.0 mmol) and (R)-tert-butyl 2-

(aminomethyl)pyrrolidine-1-carboxylate (I, 1.0 mmol) in dichloro-

methane (5 mL) was added triethylamine (140 μL, 1.0 equiv)

dropwise. The resultant was stirred at room temperature for 1 h. The

mixture was concentrated in vacuo, and the residue was dissolved in

ethyl acetate and washed with 10% NH₄Cl (aqueous) and brine (3×).

The organic phase was separated, dried over Na₂SO₄, and

concentrated. The residue was treated with 20% TFA/dichloro-

methane (5 mL) at room temperature for 30 min and concentrated to

dryness to provide the desired substituted pyrrolidine amine (NR₃,

III) as a TFA salt, which was directly used in the next step of the

reaction without further puriﬁcation.

To a solution of cyanuric chloride (18.4 mg, 0.1 mmol) in

acetonitrile (4 mL) was added the ﬁrst amine (NR₂, 1.0 equiv) at 0 °C.

The mixture was stirred at the same temperature for 30 min followed

by addition of diisopropylethylamine (DIPEA, 1.0 equiv). The mixture

was continuously stirred for another 15 min. Then to this reaction

mixture were added the second amine (NR₃, 1.0 equiv) and DIPEA

(3.0 equiv). The resulting reaction mixture was warmed to room

temperature and stirred for 2 h followed by the addition of the third

amine (NR₁, 3.0−5.0 equiv) and DIPEA (3.0 equiv). The reaction

mixture was heated at 80 °C for various times as monitored by LC−

MS to ﬁnish the third amine replacement. The reaction mixture was

concentrated in vacuo and the residue was puriﬁed by preparative

HPLC (method E) to aﬀord the ﬁnal compounds (IV) in reasonable

yields (60−80%).

N-((R)-1-(4-(Benzylamino)-6-(((S)-2-oxoazepan-3-yl)amino)-

1,3,5-triazin-2-yl)pyrrolidin-3-yl)-3-ﬂuoro-4-methylbenzene-

sulfonamide (11). By use of (S)-3-aminoazepan-2-one as NR₂, (R)-

3-ﬂuoro-4-methyl-N-(pyrrolidin-3-yl)benzenesulfonamide TFA salt as

NR₃, and benzylamine as NR₁, the title compound was prepared by

following general method A. MS (ESI) m/z [M + H]⁺= 569.3, t_R=

1.53 min (HPLC method A but with 40−98% MeCN/H₂O (+0.1%

1

TFA) over 3.5 min gradient). H NMR (400 MHz, CD₃OD) δ 7.60

(d, J = 8.0 Hz, 1H), 7.54 (m, 1H), 7.44 (m, 2H), 7.32 (m, 4H), 4.65

(m, 2H), 4.58 (m, 1H), 4.50 (m, 1H), 3.91 (m, 1H), 3.7 (m, 1H), 3.61

(m, 2H), 3.45 (m, 1H), 3.24 (m, 1H), 2.34 (s, 3H), 2.06 (m, 3H), 1.87

(m, 3H), 1.53 (m, 1H), 1.42 (m, 1H). HRMS (M + H)⁺calcd for

[C₂₇H₃₄N₈O₃FS + H] 569.2459; found 569.2459.

General Method B. By use of (R)-tert-butyl 2-(aminomethyl)-

pyrrolidine-1-carboxylate as the second amine, the triazine inter-

mediate (V) was prepared using the same procedure as described in

general method A. The intermediate V was treated with 20% TFA/

dichloromethane at room temperature for 30 min and then condensed.

The residue was redissolved in dichloromethane and treated with

sulfonyl chloride (1.0 equiv) and triethylamine (3.0 equiv). The

mixture was stirred at room temperature for 2 h and then concentrated

in vacuo. The residue was puriﬁed by preparative HPLC (method E)

to aﬀord compounds (IV) in 20−65% yields.

N-({(2R)-1-[4-(Butylamino)-6-({[2-(2-thienyl)-1,3-thiazol-4-yl]-

methyl}amino)-1,3,5-triazin-2-yl]-2-pyrrolidinyl}methyl)-4-

(triﬂuoromethyl)benzenesulfonamide (12a). By use of {[2-(2-

thienyl)-1,3-thiazol-4-yl]methyl}amine as NR₂, N-[(2R)-2-pyrrolidi-

nylmethyl]-4-(triﬂuoromethyl)benzenesulfonamide TFA salt as NR₃,

and n-butylamine as NR₃, the title compound was prepared by

following general method A. MS (ESI) m/z [M + 1]⁺= 653.2, t_R=

N-({(2R)-1-[4-(3-Buten-1-ylamino)-6-({[2-(2-thienyl)-1,3-thia-

zol-4-yl]methyl}amino)-1,3,5-triazin-2-yl]-2-pyrrolidinyl}-

methyl)-4-(triﬂuoromethyl)benzenesulfonamide (7). By use of

{[2-(2-thienyl)-1,3-thiazol-4-yl]methyl}amine as NR₂, N-[(2R)-2-

pyrrolidinylmethyl]-4-(triﬂuoromethyl)benzenesulfonamide TFA salt

as NR₃, and 3-buten-1-amine as NR₁, the title compound was prepared

by following general method A. MS (ESI) m/z [M + H]⁺= 651.2, t_R=

1

7.84 min (HPLC method B); H NMR (400 MHz, CD₃OD) δ 7.99

(d, J = 8.0 Hz, 1H), 7.91 (m, 1H), 7.87 (d, J = 8.4 Hz, 1H), 7.76 (d, J

= 8.4 Hz, 1H), 7.60 (d, J = 3.6 Hz, 1H), 7.56 (d, J = 5.6 Hz, 1H), 7.36

(s, 1H), 7.12 (t, J = 4.0 Hz, 1H), 4.82−4.68 (m, 3H), 4.27 (br, 1H),

3.65−3.35 (m, 3H), 3.25−3.05 (m, 3H), 2.06−1.92 (m, 4H), 1.62−

1.28 (m, 5H), 0.98−0.86 (m, 3H). HRMS (M + H)⁺calcd for

[C₂₇H₃₁F₃N₈O₂S₃+ H] 653.1757; found 653.1717.

1

7.55 min (HPLC method B); H NMR (400 MHz, CD₃OD) δ 7.99

N-({(2R)-1-[4-[(Cyclopropylmethyl)amino]-6-({[2-(2-thienyl)-

1,3-thiazol-4-yl]methyl}amino)-1,3,5-triazin-2-yl]-2-

pyrrolidinyl}methyl)-4-(triﬂuoromethyl)benzenesulfonamide

(12b). By use of {[2-(2-thienyl)-1,3-thiazol-4-yl]methyl}amine as

NR₂, N-[(2R)-2-pyrrolidinylmethyl]-4-(triﬂuoromethyl)-

benzenesulfonamide TFA salt as NR₃, and 1-cyclopropylmethanamine

as NR₁, the title compound was prepared by following general method

A. MS (ESI) m/z [M + 1]⁺= 651.3, t_R= 7.42 min (HPLC method B);

¹H NMR (400 MHz, CD₃OD) δ 7.70 (d, J = 8.4 Hz, 1H), 7.57 (m,

(d, J = 8.0 Hz, 1H), 7.91 (m, 1H), 7.87 (d, J = 8.4 Hz, 1H), 7.76 (d, J

= 8.4 Hz, 1H), 7.60 (d, J = 3.6 Hz, 1H), 7.56 (d, J = 5.6 Hz, 1H), 7.36

(s, 1H), 7.12 (t, J = 4.0 Hz, 1H), 5.80 (m, 1H), 5.07 (m, 2H), 4.78−

4.68 (m, 3H), 4.26 (br, 1H), 3.65−3.47 (m, 3H), 3.25−3.05 (m, 3H),

2.38−2.28 (m, 2H), 2.10−1.86 (m, 5H). HRMS (M + H)⁺calcd for

[C₂₇H₂₉F₃N₈O₂S₃+ H] 651.1600; found 651.1577.

N-({(2R)-1-[4-(3-Buten-1-ylamino)-6-({[2-(2-thienyl)-1,3-thia-

zol-4-yl]methyl}amino)-1,3,5-triazin-2-yl]-2-pyrrolidinyl}-

methyl)-4-propylbenzenesulfonamide (8). By use of {[2-(2-

thienyl)-1,3-thiazol-4-yl]methyl}amine as NR₂, 4-propyl-N-[(2R)-2-

pyrrolidinylmethyl]benzenesulfonamide TFA salt as NR₃, and 3-

buten-1-amine as NR₁, the title compound was prepared by following

2H), 7.46 (d, J = 7.6 Hz, 1H), 7.29 (m, 2H), 7.07 (s, 1H), 6.83 (m,

1H), 4.40 (m, 2H), 3.96 (m, 1H), 3.30 (m, 2H), 2.97 (m, 1H), 2.87−

2.83 (m, 2H), 1.76 (m, 3H), 1.62 (m, 1H), 0.81−0.74 (m, 2H), 0.26−

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0.19 (m, 2H), 0.00 (m, 2H). HRMS (M + H)⁺calcd for

[C₂₇H₂₉F₃N₈O₂S₃+ H] 651.1600; found 651.1580.

methylpropan-1-amine as NR₁, the title compound was prepared by

following general method A. MS (ESI) m/z [M + 1]⁺= 621.4, t_R=

8.11 min (HPLC method B). HRMS (M + H)⁺calcd for

[C₃₁H₄₀N₈O₂S₂+ H] 621.2788; found 621.2763.

N-{[(2R)-1-(4-[(2-Methylpropyl)amino]-6-{[(2-phenyl-1,3-

thiazol-4-yl)methyl]amino}-1,3,5-triazin-2-yl)-2-pyrrolidinyl]-

methyl}-4-propylbenzenesulfonamide (13c). By use of (2-

phenyl-1,3-thiazol-4-yl)methanamine as NR₂, 4-propyl-N-[(2R)-2-

pyrrolidinylmethyl]benzenesulfonamide TFA salt as NR₃, and 2-

methylpropan-1-amine as NR₁, the title compound was prepared by

following general method A. LC−MS (ESI) m/z [M + 1]⁺= 621.4, t_R

= 8.36 min (HPLC method B). HRMS (M + H)⁺calcd for

[C₃₁H₄₀N₈O₂S₂+ H] 621.2788; found 621.2749.

N-{[(2R)-1-(4-[(2-Cyclopropylethyl)amino]-6-{[(2-phenyl-1,3-

thiazol-4-yl)methyl]amino}-1,3,5-triazin-2-yl)-2-pyrrolidinyl]-

methyl}-4-propylbenzenesulfonamide (13d). By use of (2-

phenyl-1,3-thiazol-4-yl)methanamine as NR₂, 4-propyl-N-[(2R)-2-

pyrrolidinylmethyl]benzenesulfonamide TFA salt as NR₃, and 2-

cyclopropylethanamine as NR₁, the title compound was prepared by

following general method A. LC−MS (ESI) m/z [M + 1]⁺= 633.4, t_R

= 8.37 min (HPLC method B). HRMS (M + H)⁺calcd for

[C₃₂H₄₀N₈O₂S₂+ H] 633.2788; found 633.2753.

N-{[(2R)-1-(4-(Cyclopropylamino)-6-{[(2-phenyl-1,3-thiazol-

4-yl)methyl]amino}-1,3,5-triazin-2-yl)-2-pyrrolidinyl]methyl}-

4-propylbenzenesulfonamide (13e). By use of (2-phenyl-1,3-

thiazol-4-yl)methanamine as NR₂, 4-propyl-N-[(2R)-2-

pyrrolidinylmethyl]benzenesulfonamide TFA salt as NR₃, and cyclo-

propanamine as NR₁, the title compound was prepared by following

general method A. LC−MS (ESI) m/z [M + 1]⁺= 605.3, t_R= 7.26

min (HPLC method B). HRMS (M + H)⁺calcd for [C₃₀H₃₆N₈O₂S₂+

H] 605.2475; found 605.2440.

N-({(2R)-1-[4-(Methylamino)-6-({[2-(2-thienyl)-1,3-thiazol-4-

yl]methyl}amino)-1,3,5-triazin-2-yl]-2-pyrrolidinyl}methyl)-4-

(triﬂuoromethyl)benzenesulfonamide (12c). By use of {[2-(2-

thienyl)-1,3-thiazol-4-yl]methyl}amine as NR₂, N-[(2R)-2-pyrrolidi-

nylmethyl]-4-(triﬂuoromethyl)benzenesulfonamide TFA salt as NR₃,

and methylamine as NR₁, the title compound was prepared by

following general method A. MS (ESI) m/z [M + 1]⁺= 611.16, t_R=

1

6.27 min (HPLC method B). H NMR (400 MHz, CD₃OD) δ 7.99

(d, J = 8.4 Hz, 1H), 7.87 (m, 2H), 7.75 (d, J = 8.4 Hz, 1H), 7.59 (d, J

= 3.6 Hz, 1H), 7.55 (d, J = 4.8 Hz, 1H), 7.35 (s, 1H), 7.12 (t, J = 4.4

Hz, 1H), 4.81−4.77 (m, 1H), 4.67 (m, 1H), 4.26 (m 1H), 3.66−3.58

(m, 2H), 3.27−3.11 (m, 2H), 2.94−2.90 (d, J = 16.4 Hz, 2H), 2.05

(m, 3H), 1.91 (m, 1H). HRMS (M + H)⁺calcd for [C₂₄H₂₅F₃N₈O₂S₃

+ H] 611.1287; found 611.1277.

N-({(2R)-1-[4-Oxo-6-({[2-(2-thienyl)-1,3-thiazol-4-yl]methyl}-

amino)-1,4-dihydro-1,3,5-triazin-2-yl]-2-pyrrolidinyl}methyl)-

4-(triﬂuoromethyl)benzenesulfonamide (12d). To a solution of

cyanuric chloride (18.4 mg, 0.1 mmol) in acetonitrile (4 mL) was

added {[2-(2-thienyl)-1,3-thiazol-4-yl]methyl}amine (1.0 equiv) at 0

°C. The mixture was stirred at the same temperature for 30 min

followed by addition of DIPEA (3.0 equiv). The mixture was

continuously stirred for another 15 min. Then to this reaction mixture

was added N-[(2R)-2-pyrrolidinylmethyl]-4-(triﬂuoromethyl)-

benzenesulfonamide TFA salt (1.0 equiv). The resulting reaction

mixture was warmed to room temperature and stirred for 2 h. The

mixture was then treated with CH₃CN/TFA/H₂O/anisole (2 mL,

50:40:10:1, v/v/v/v) at room temperature overnight. The reaction

mixture was concentrated in vacuo and the residue was puriﬁed by

preparative HPLC (method E) to aﬀord the title compound (27 mg,

45% yield). MS (ESI) m/z [M + 1]⁺= 598.2, t_R= 4.85 min (HPLC

method B). ¹H NMR (400 MHz, CD₃OD) δ 7.88 (d, J = 8.4 Hz, 2H),

7.76 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 5.2 Hz, 1H), 7.54 (dd, J = 5.2, 0.8

Hz, 1H), 7.35 (s, 1H), 7.13 (dd, J = 4.8, 4.0 Hz, 1H), 4.81 (s, 2H),

4.71 (d, J = 15.6 Hz, 1H), 4.42 (m, 1H), 3.64−3.51 (m, 2H), 3.27 (d, J

= 3.2 Hz, 1H), 3.16 (dd, J = 7.2, 14.0 Hz, 1H), 2.20 (m, 1H), 2.1 (m,

2H). HRMS (M + H)⁺calcd for [C₂₃H₂₂F₃N₇O₃S₃+ H] 598.0971;

found 598.0943.

N-{[(2R)-1-(4-(Cyclopentylamino)-6-{[(2-phenyl-1,3-thiazol-

4-yl)methyl]amino}-1,3,5-triazin-2-yl)-2-pyrrolidinyl]methyl}-

4-propylbenzenesulfonamide (13f). By use of (2-phenyl-1,3-

thiazol-4-yl)methanamine as NR₂, 4-propyl-N-[(2R)-2-

pyrrolidinylmethyl]benzenesulfonamide TFA salt as NR₃, and cyclo-

pentanamine as NR₁, the title compound was prepared by following

general method A. LC−MS (ESI) m/z [M + 1]⁺= 633.4, t_R= 8.55

min (HPLC method B). HRMS (M + H)⁺calcd for [C₃₂H₄₁N₈O₂S₂+

H] 633.2794; found 633.2793.

N-({(2R)-1-[4-({[2-(2-Thienyl)-1,3-thiazol-4-yl]methyl}amino)-

1,3,5-triazin-2-yl]-2-pyrrolidinyl}methyl)-4-(triﬂuoromethyl)-

benzenesulfonamide (12e). To a solution of 2,4-dichloro-1,3,5-

triazine (5.5 mg, 0.05 mmol) in CH₃CN−THF (2:1, 3 mL) was added

{[2-(2-thienyl)-1,3-thiazol-4-yl]methyl}amine (10 mg, 0.05 mmol).

The mixture was stirred at room temperature for 45 min. Then N-

[(2R)-2-pyrrolidinylmethyl]-4-(triﬂuoromethyl)benzenesulfonamide

TFA salt (0.05 mmol) and DIPEA (0.15 mmol) were added. The

resultant was heated at 60 °C overnight. The reaction mixture was

condensed in vacuo and the residue was puriﬁed by preparative HPLC

(method E) to aﬀord the title compound (10 mg, 34% yield). MS

N-{[(2R)-1-(4-[(2-Cyanoethyl)amino]-6-{[(2-phenyl-1,3-thia-

zol-4-yl)methyl]amino}-1,3,5-triazin-2-yl)-2-pyrrolidinyl]-

methyl}-4-propylbenzenesulfonamide (13g). By use of (2-

phenyl-1,3-thiazol-4-yl)methanamine as NR₂, 4-propyl-N-[(2R)-2-

pyrrolidinylmethyl]benzenesulfonamide TFA salt as NR₃, and 3-

aminopropanenitrile as NR₁, the title compound was prepared by

following general method A. LC−MS (ESI) m/z [M + 1]⁺= 618.3, t_R

1

= 6.73 min (HPLC method B); H NMR (400 MHz, CD₃OD) δ

7.96−7.90 (m, 2H), 7.72−7.60 (m, 2H), 7.43−7.42 (br, 3H), 7.3−7.10

(br, 3H), 4.80 (m, 2H), 3.80−3.40 (m, 4H), 3.30−2.90 (m, 2H),

2.70−2.58 (m, 4H), 2.10−1.25 (br, 7H), 0.90−0.94 (t, 3H). HRMS

(M + H)⁺calcd for [C₃₀H₃₅N₉O₂S₂+ H] 618.2428; found 618.2403.

N-{[(2R)-1-(4-[(2-Hydroxyethyl)amino]-6-{[(2-phenyl-1,3-

thiazol-4-yl)methyl]amino}-1,3,5-triazin-2-yl)-2-pyrrolidinyl]-

methyl}-4-propylbenzenesulfonamide (13h). By use of (2-

phenyl-1,3-thiazol-4-yl)methanamine as NR₂, 4-propyl-N-[(2R)-2-

pyrrolidinylmethyl]benzenesulfonamide TFA salt as NR₃, and 2-

aminoethanol as NR₁, the title compound was prepared by following

general method A. LC−MS (ESI) m/z [M + 1]⁺= 609.3, t_R= 6.03

min (HPLC method B). HRMS (M + H)⁺calcd for [C₂₉H₃₆N₈O₃S₂+

H] 609.2425; found 609.2397.

N-{[(2R)-1-(4-{[2-(Methyloxy)ethyl]amino}-6-{[(2-phenyl-1,3-

thiazol-4-yl)methyl]amino}-1,3,5-triazin-2-yl)-2-pyrrolidinyl]-

methyl}-4-propylbenzenesulfonamide (13i). By use of (2-phenyl-

1,3-thiazol-4-yl)methanamine as NR₂, 4-propyl-N-[(2R)-2-

pyrrolidinylmethyl]benzenesulfonamide TFA salt as NR₃, and 2-

methoxyethanamine as NR₁, the title compound was prepared by

following general method A. LC−MS (ESI) m/z [M + 1]⁺= 623.4, t_R

= 6.89 min (HPLC method B). HRMS (M + H)⁺calcd for

[C₃₀H₃₈N₈O₃S₂+ H] 623.2581; found 623.2547.

1

(ESI) m/z [M + 1]⁺= 582.1, t_R= 5.49 min (HPLC method B). H

NMR (400 MHz, CD₃OD) δ 8.23 (s, 1H), 7.88 (d, J = 8.0 Hz, 2H),

7.75 (d, J = 8.0 Hz, 2H), 7.55 (d, J = 2.4 Hz, 1H), 7.53 (d, J = 2.4 Hz,

1H), 7.34 (s, 1H), 7.12 (t, J = 4.4 Hz, 1H), 4.73 (d, J = 9.2 Hz, 2H),

4.34 (m, 1H), 3.65 (t, J = 5.4 Hz, 2H), 3.17 (m, 2H), 2.17−2.05 (m,

3H), 2.01 (m, 1H). HRMS (M + H)⁺calcd for [C₂₃H₂₂F₃N₇O₂S₃+

H] 582.1022; found 582.1019.

N-({(2R)-1-[4-{[(2-Phenyl-1,3-thiazol-4-yl)methyl]amino}-6-

(propylamino)-1,3,5-triazin-2-yl]-2-pyrrolidinyl}methyl)-4-pro-

pylbenzenesulfonamide (13a). By use of (2-phenyl-1,3-thiazol-4-

yl)methanamine as NR₂, 4-propyl-N-[(2R)-2-pyrrolidinylmethyl]-

benzenesulfonamide TFA salt as NR₃, and propylamine as NR₁, the

title compound was prepared by following general method A. LC−MS

(ESI) m/z [M + 1]⁺= 607.4, t_R= 7.76 min (HPLC method B).

HRMS (M + H)⁺calcd for [C₃₀H₃₈N₈O₂S₂+ H] 607.2632; found

607.2593.

N-{[(2R)-1-(4-[Methyl(propyl)amino]-6-{[(2-phenyl-1,3-thia-

zol-4-yl)methyl]amino}-1,3,5-triazin-2-yl)-2-pyrrolidinyl]-

methyl}-4-propylbenzenesulfonamide (13b). By use of (2-

phenyl-1,3-thiazol-4-yl)methanamine as NR₂, 4-propyl-N-[(2R)-2-

pyrrolidinylmethyl]benzenesulfonamide TFA salt as NR₃, and N-

7073

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Journal of Medicinal Chemistry

Article

N-[((2R)-1-{4-{[(2-Phenyl-1,3-thiazol-4-yl)methyl]amino}-6-

[(tetrahydro-2H-pyran-4-ylmethyl)amino]-1,3,5-triazin-2-yl}-2-

pyrrolidinyl)methyl]-4-propylbenzenesulfonamide (13j). By

use of (2-phenyl-1,3-thiazol-4-yl)methanamine as NR₂, 4-propyl-N-

[(2R)-2-pyrrolidinylmethyl]benzenesulfonamide TFA salt as NR₃, and

(tetrahydro-2H-pyran-4-yl)methanamine as NR₁, the title compound

was prepared by following general method A. LC−MS (ESI) m/z [M

+ 1]⁺= 663.4, t_R= 7.13 min (HPLC method B). HRMS (M + H)⁺

calcd for [C₃₃H₄₃N₈O₃S₂+ H] 663.2900; found 663.2900.

N-{[(2R)-1-(4-(3-Buten-1-ylamino)-6-{[(2-phenyl-1,3-thiazol-

4-yl)methyl]amino}-1,3,5-triazin-2-yl)-2-pyrrolidinyl]methyl}-

4-(triﬂuoromethyl)benzenesulfonamide (14a). By use of (2-

phenyl-1,3-thiazol-4-yl)methanamine as NR₂, N-[(2R)-2-pyrrolidinyl-

methyl]-4-(triﬂuoromethyl)benzenesulfonamide TFA salt as NR₃, and

3-buten-1-amine as NR₁, the title compound was prepared by

following general method A. MS (ESI) m/z [M + 1]⁺= 645.3, t_R=

7.81 min (HPLC method B). HRMS (M + H)⁺calcd for

[C₂₉H₃₁F₃N₈O₂S₂+ H] 645.2036; found 645.1996.

zenesulfonamide (15b). By use of (4-methylthiophen-2-yl)-

methanamine as NR₂, 4-propyl-N-[(2R)-2-pyrrolidinylmethyl]-

benzenesulfonamide TFA salt as NR₃, and n-butylamine as NR₁, the

title compound was prepared by following general method A. MS

1

(ESI) m/z [M + 1]⁺= 558.3, t_R= 1.62 min (HPLC method D); H

NMR (CDCl₃, 300 MHz) δ 7.90 (s, 1 H), 7.80 (s, 1 H), 7.68−7.64

(m, 3H), 7.27−7.24 (m, 2 H), 6.80−6.78 (m, 1 H), 6.75 (s, 1 H), 4.70

(m, 2 H), 4.31 (m, 1 H), 3.68−3.16 (m, 6 H), 2.64−2.61 (m, 2 H),

2.20 (m, 3 H), 1.99 (m, 4 H), 1.67−1.56 (m, 4 H),1.36−1.33 (m, 2

H), 0.94−0.89 (t, 6 H). HRMS (M + H)⁺calcd for [C₂₇H₃₉N₇O₂S₂+

H] 558.2679; found 558.2671.

N-{[(2R)-1-(4-(Butylamino)-6-{[(4-cyano-2-thienyl)methyl]-

amino}-1,3,5-triazin-2-yl)-2-pyrrolidinyl]methyl}-4-propylben-

zenesulfonamide (15c). By use of 5-(aminomethyl)thiophene-3-

carbonitrile as NR₂, 4-propyl-N-[(2R)-2-pyrrolidinylmethyl]-

benzenesulfonamide TFA salt as NR₃, and n-butylamine as NR₁, the

title compound was prepared by following general method A. MS

1

(ESI) m/z [M + 1]⁺= 569.2, t_R= 1.56 min (HPLC method D); H

N-({(2R)-1-[4-[(4-Biphenylylmethyl)amino]-6-(3-buten-1-yla-

mino)-1,3,5-triazin-2-yl]-2-pyrrolidinyl}methyl)-4-

(triﬂuoromethyl)benzenesulfonamide (14b). By use of (4-

biphenylylmethyl)amine as NR₂, N-[(2R)-2-pyrrolidinylmethyl]-4-

(triﬂuoromethyl)benzenesulfonamide TFA salt as NR₃, and 3-buten-

1-amine as NR₁, the title compound was prepared by following general

method A. MS (ESI) m/z [M + 1]⁺= 638.3, t_R= 8.60 min (HPLC

method B). HRMS (M + H)⁺calcd for [C₃₂H₃₄F₃N₇O₂S + H]

638.2520; found 638.2503.

N-({(2R)-1-[4-[(3-Biphenylylmethyl)amino]-6-(3-buten-1-yla-

mino)-1,3,5-triazin-2-yl]-2-pyrrolidinyl}methyl)-4-

(triﬂuoromethyl)benzenesulfonamide (14c). By use of (3-

biphenylylmethyl)amine as NR₂, N-[(2R)-2-pyrrolidinylmethyl]-4-

(triﬂuoromethyl)benzenesulfonamide TFA salt as NR₃, and 3-buten-

1-amine as NR₁, the title compound was prepared by following general

method A. MS (ESI) m/z [M + 1]⁺= 638.3, t_R= 8.60 min (HPLC

method B). HRMS (M + H)⁺calcd for [C₃₂H₃₄F₃N₇O₂S + H]

638.2520; found 638.2486.

NMR (CDCl₃, 300 MHz) δ 8.50 (s, 1 H), 8.60 (s, 1 H), 7.81 (s, 1 H),

7.73−7.67 (m, 3 H), 7.29−7.25 (m, 2 H), 7.19−7.18 (m, 1 H), 4.89

(m, 2 H), 4.31 (m, 1 H), 3.70−3.20 (m, 6 H), 2.98 (m, 1 H), 2.65−

2.62 (m, 2 H), 2.0−1.97 (m, 5 H), 1.67−1.56 (m, 4 H),1.36−1.33 (m,

2 H), 0.94−0.89 (t, 6 H). HRMS (M + H)⁺calcd for [C₂₇H₃₆N₈O₂S₂

+ H] 569.2475; found 569.2468.

N-{[(2R)-1-(4-(Butylamino)-6-{[(6-methyl-2-pyridinyl)-

methyl]amino}-1,3,5-triazin-2-yl)-2-pyrrolidinyl]methyl}-4-pro-

pylbenzenesulfonamide (15d). By use of 1-(6-methyl-2-pyridinyl)-

methanamine as NR₂, 4-propyl-N-[(2R)-2-pyrrolidinylmethyl]-

benzenesulfonamide TFA salt as NR₃, and n-butylamine as NR₁, the

title compound was prepared by following general method A. MS

1

(ESI) m/z [M + 1]⁺= 553.3, t_R= 1.40 min (HPLC method D); H

NMR (CDCl₃, 300 MHz) δ 9.18 (s, 1 H), 8.10 (t, 1 H), 7.72−7.70

(m, 3 H), 7.50 (m, 1 H), 7.27 (m, 3 H), 6.89 (m, 1 H), 5.24 (m, 2 H),

4.21 (m, 1 H), 3.52−3.48 (m, 4 H), 2.87 (m, 1 H), 2.86 (s, 3 H), 2.85

(m, 2 H), 2.65−2.60 (m, 2 H), 1.97 (m, 3 H), 1.67−1.56 (m, 4 H),

1.36−1.33 (m, 2 H), 0.94−0.89 (t, 6 H). HRMS (M + H)⁺calcd for

[C₂₈H₄₀N₈O₂S + H] 553.3068; found 553.3086.

N-{[(2R)-1-(4-(3-Buten-1-ylamino)-6-{[(2-methyl-1,3-thiazol-

4-yl)methyl]amino}-1,3,5-triazin-2-yl)-2-pyrrolidinyl]methyl}-

4-(triﬂuoromethyl)benzenesulfonamide (14d). By use of 1-(2-

methyl-1,3-thiazol-4-yl)methanamine as NR₂, N-[(2R)-2-pyrrolidinyl-

methyl]-4-(triﬂuoromethyl)benzenesulfonamide TFA salt as NR₃, and

3-buten-1-amine as NR₁, the title compound was prepared by

following general method A. MS (ESI) m/z [M + 1]⁺= 583.2, t_R=

N-{[(2R)-1-(4-(Butylamino)-6-{[(3-methylphenyl)methyl]-

amino}-1,3,5-triazin-2-yl)-2-pyrrolidinyl]methyl}-4-propylben-

zenesulfonamide (15e). By use of 1-(3-methylphenyl)methanamine

as NR₂, 4-propyl-N-[(2R)-2-pyrrolidinylmethyl]benzenesulfonamide

TFA salt as NR₃, and n-butylamine as NR₁, the title compound was

prepared by following general method A. MS (ESI) m/z [M + 1]⁺=

1

5.74 min (HPLC method B). H NMR (400 MHz, CD₃OD) δ 7.99

552.3, t_R= 1.63 min (HPLC method D); H NMR (CDCl₃, 300

(d, J = 8.0 Hz, 2H), 7.91 (d, J = 7.2 Hz, 1H), 7.85 (m, 2H), 7.08 (s,

1H), 5.82 (m, 1H), 5.13−5.00 (m, 2H), 4.82 (m, 2H), 4.25 (m, 1H),

3.66−3.46 (m, 4H), 3.26−3.02 (m, 2H), 2.39 (s, 3H), 2.34 (m, 2H),

2.06−1.91 (m, 4H). HRMS (M + H)⁺calcd for [C₂₄H₂₉F₃N₈O₂S₂+

H] 583.1880; found 583.1856.

MHz) δ 7.89 (s, 1 H), 7.67 (m, 3 H), 7.26 (m, 3 H), 7.08 (m, 2 H),

4.53 (m, 2 H), 3.63−3.36 (m, 4 H), 3.0 (m, 2 H), 2.64−2.59 (m, 2 H),

2.34 (s, 3 H), 2.20 (m, 1 H), 1.94 (m, 4 H), 1.67−1.56 (m, 4 H),

1.36−1.33 (m, 2 H), 0.94−0.89 (t, 6 H). HRMS (M + H)⁺calcd for

[C₂₉H₄₁N₇O₂S + H] 552.3115; found 552.3088.

N-[((2R)-1-{4-(3-Buten-1-ylamino)-6-[(phenylmethyl)amino]-

1,3,5-triazin-2-yl}-2-pyrrolidinyl)methyl]-4-(triﬂuoromethyl)-

benzenesulfonamide (14e). By use of benzylamine as NR₂, N-

[(2R)-2-pyrrolidinylmethyl]-4-(triﬂuoromethyl)benzenesulfonamide

TFA salt as NR₃, and 3-buten-1-amine as NR₁, the title compound was

prepared by following general method A. MS (ESI) m/z [M + 1]⁺=

562.3, t_R= 7.48 min (HPLC method B). HRMS (M + H)⁺calcd for

[C₂₆H₃₀F₃N₇O₂S + H] 562.2207; found 562.2193.

N-[((2R)-1-{4-(Butylamino)-6-[(3-thienylmethyl)amino]-1,3,5-

triazin-2-yl}-2-pyrrolidinyl)methyl]-4-propylbenzenesulfona-

mide (15a). By use of thiophen-3-ylmethanamine as NR₂, 4-propyl-

N-[(2R)-2-pyrrolidinylmethyl]benzenesulfonamide TFA salt as NR₃,

and n-butylamine as NR₁, the title compound was prepared by

following general method A. MS (ESI) m/z [M + 1]⁺= 544.3, t_R=

1.60 min (HPLC method D); ¹H NMR (CDCl₃, 300 MHz) δ 8.07 (s,

1 H), 7.68−7.63 (m, 3 H), 7.25−7.18 (m, 3 H), 7.06−7.04 (m, 1 H),

6.08 (s, 1 H), 5.96 (s, 1 H), 4.55−4.53 (m, 2 H), 4.28−4.10 (m,1 H),

3.46−3.32 (m, 6 H), 2.63−2.58 (m, 2 H), 1.94 (m, 4 H), 1.67−1.56

(m, 4 H),1.36−1.33 (m, 2 H), 0.95−0.89 (t, 6 H). HRMS (M + H)⁺

calcd for [C₂₆H₃₇N₇O₂S₂+ H] 544.2523; found 544.2498.

N-{[(2R)-1-(4-(Butylamino)-6-{[(1-methyl-1H-imidazol-4-yl)-

methyl]amino}-1,3,5-triazin-2-yl)-2-pyrrolidinyl]methyl}-4-pro-

pylbenzenesulfonamide (15f). By use of (1-methyl-1H-imidazol-4-

yl)methanamine as NR₂, 4-propyl-N-[(2R)-2-pyrrolidinylmethyl]-

benzenesulfonamide TFA salt as NR₃, and n-butylamine as NR₁, the

title compound was prepared by following general method A. MS

1

(ESI) m/z [M + 1]⁺= 542.3, t_R= 1.27 min (HPLC method D); H

NMR (CDCl₃, 300 MHz) δ 8.02 (s, 1 H), 7.61−7.58 (d, 2 H), 7.35 (s,

1 H), 7.21−7.18 (d, 2 H), 6.92−6.78 (m, 1 H), 5.0 (s, 1 H), 5.48 (s, 1

H), 4.50 (m, 2 H), 3.63 (t, 3 H), 3.40−2.8 (m, 6 H),1.90−1.00 (m, 11

H), 0.94−0.89 (t, 6 H). HRMS (M + H)⁺calcd for [C₂₆H₃₉N₉O₂S +

H] 542.3020; found 542.3023.

N-{[(2R)-1-(4-(Butylamino)-6-{[(4-butylphenyl)methyl]-

amino}-1,3,5-triazin-2-yl)-2-pyrrolidinyl]methyl}-4-propylben-

zenesulfonamide (15g). By use of (4-butylphenyl)methanamine as

NR₂, 4-propyl-N-[(2R)-2-pyrrolidinylmethyl]benzenesulfonamide

TFA salt as NR₃, and n-butylamine as NR₁, the title compound was

prepared by following general method A. MS (ESI) m/z [M + 1]⁺=

1

594.4, t_R= 1.74 min (HPLC method D); H NMR (CDCl₃, 300

MHz) δ 7.80 (s, 1 H), 7.67−7.61 (m, 2 H), 7.24−7.12 (m, 6 H), 5.70

(s, 1 H), 5.40 (s, 1 H), 4.53−4.10 (m, 3 H), 3.62−2.90 (m, 6 H),

2.64−2.56 (m, 4 H), 1.94−1.90 (m, 4 H), 1.62−1.52 (m, 6 H), 1.36−

N-{[(2R)-1-(4-(Butylamino)-6-{[(4-methyl-2-thienyl)methyl]-

amino}-1,3,5-triazin-2-yl)-2-pyrrolidinyl]methyl}-4-propylben-

7074

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Journal of Medicinal Chemistry

Article

1.33 (m, 4 H), 0.94−0.89 (t, 9 H). HRMS (M + H)⁺calcd for

[C₃₂H₄₈N₇O₂S + H] 594.3590; found 594.3589.

and 3-buten-1-amine as NR₁, the title compound was prepared by

following general method A. MS (ESI) m/z [M + 1]⁺= 645.3, t_R=

7.64 min (HPLC method B). ¹H NMR (400 MHz, DMSO-d₆) δ 8.10

(m, 2H), 7.96 (m, 4H), 7.91 (d, J = 2.8 Hz, 1H), 7.89 (s, 1H), 7.48 (d,

J = 5.2 Hz, 2H), 7.47 (s 1H), 5.78 (m, 1H), 5.05 (m, 2H), 4.65 (s,

2H), 3.82 (br, 2H), 3.62 (m, 2H), 3.45−3.34 (m, 3H), 3.20 (m 1H),

2.83 (t, J = 5.2, 2H), 2.28 (m, 2H), 2.18 (m, 1H), 1.96 (m, 1H), 1.65

(m, 1H). HRMS (M + H)⁺calcd for [C₂₉H₃₁F₃N₈O₂S₂+ H]

645.2036; found 645.2018.

N-({(2R)-1-[4-(Butylamino)-6-(cyclopentylamino)-1,3,5-tria-

zin-2-yl]-2-pyrrolidinyl}methyl)-4-propylbenzenesulfonamide

(15h). By use of cyclopentanamine as NR₂, 4-propyl-N-[(2R)-2-

pyrrolidinylmethyl]benzenesulfonamide TFA salt as NR₃, and n-

butylamine as NR₁, the title compound was prepared by following

general method A. MS (ESI) m/z [M + 1]⁺= 516.3, t_R= 1.62 min

1

(HPLC method D); H NMR (CDCl₃, 300 MHz) δ 7.70−7.67 (d, 2

H), 7.30−7.29 (d, 2 H), 7.50 (s, 1 H), 7.27 (s, 1 H), 5.75 (s, 1 H),

4.35−4.20 (m, 2 H), 3.67 (m, 1 H), 3.39 (m, 2 H), 3.20 (m,1 H), 2.90

(m, 1 H), 2.65−2.63 (m, 2 H), 2.03−1.92 (m, 6 H), 1.76−1.36 (m, 11

H), 1.36 (m, 2 H), 0.98−0.92 (t, 6 H). HRMS (M + H)⁺calcd for

[C₂₆H₄₁N₇O₂S + H] 516.3115; found 516.3093.

N-{[(2R)-1-(4-(3-Buten-1-ylamino)-6-{[(2-phenyl-1,3-thiazol-

4-yl)methyl]amino}-1,3,5-triazin-2-yl)-2-pyrrolidinyl]methyl}-

2-(triﬂuoromethyl)benzenesulfonamide (19a). By following

general method B, (R)-6-(2-(aminomethyl)pyrrolidin-1-yl)-N2-(but-

3-en-1-yl)-N4-((2-phenylthiazol-4-yl)methyl)-1,3,5-triazine-2,4-dia-

mine was ﬁrst made, which was then treated with 3-(triﬂuoromethyl)-

benzene-1-sulfonyl chloride to aﬀord the title compound in 36% yield.

LC−MS (ESI) m/z [M + 1]⁺= 645.3, t_R= 9.66 min (HPLC method

C). HRMS (M + H)⁺calcd for [C₂₉H₃₁F₃N₈O₂S₂+ H] 645.2036;

found 645.2011.

N-{[(2R)-1-(4-(3-Buten-1-ylamino)-6-{[(2-phenyl-1,3-thiazol-

4-yl)methyl]amino}-1,3,5-triazin-2-yl)-2-pyrrolidinyl]methyl}-

2-(triﬂuoromethyl)benzenesulfonamide (19b). By following

general method B, (R)-6-(2-(aminomethyl)pyrrolidin-1-yl)-N2-(but-

3-en-1-yl)-N4-((2-phenylthiazol-4-yl)methyl)-1,3,5-triazine-2,4-dia-

mine was ﬁrst made, which was then treated with 2-(triﬂuoromethyl)-

benzene-1-sulfonyl chloride to aﬀord the title compound in 39% yield.

LC−MS (ESI) m/z [M + 1]⁺= 645.3, t_R= 9.32 min (HPLC method

C). HRMS (M + H)⁺calcd for [C₂₉H₃₁F₃N₈O₂S₂+ H] 645.2036;

found 645.2007.

N-[((2R)-1-{4-(Butylamino)-6-[(cyclopropylmethyl)amino]-

1,3,5-triazin-2-yl}-2-pyrrolidinyl)methyl]-4-propylbenzenesul-

fonamide (15i). By use of cyclopropylmethanamine as NR₂, 4-

propyl-N-[(2R)-2-pyrrolidinylmethyl]benzenesulfonamide TFA salt as

NR₃, and n-butylamine as NR₁, the title compound was prepared by

following general method A. MS (ESI) m/z [M + 1]⁺= 502.3, t_R=

1.57 min (HPLC method D); ¹H NMR (CDCl₃, 300 MHz) δ 7.70 (s,

1 H), 7.68−7.66 (d, 2 H), 7.45 (s, 1 H), 7.27−7.25 (d, 2 H), 5.75 (s, 1

H), 4.35 (m, 1 H), 3.38−3.17 (m, 6 H), 2.66−2.61 (m, 2 H), 2.00−

1.91 (m, 3 H), 1.66−1.56 (m, 4 H), 1.37−1.35 (m, 2 H), 1.09 (m, 1

H), 0.96−0.89 (t, 6 H), 0.57−0.53 (m, 2 H), 0.27−0.24 (m, 2 H).

HRMS (M + H)⁺calcd for [C₂₅H₃₉N₇O₂S + H] 502.2959; found

502.2932.

N-[((2R)-1-{4-(Butylamino)-6-[(1-methylethyl)amino]-1,3,5-

triazin-2-yl}-2-pyrrolidinyl)methyl]-4-propylbenzenesulfona-

mide (15j). By use of isopropylamine as NR₂, 4-propyl-N-[(2R)-2-

pyrrolidinylmethyl]benzenesulfonamide TFA salt as NR₃, and n-

butylamine as NR₁, the title compound was prepared by following

general method A. MS (ESI) m/z [M + 1]⁺= 490.3, t_R= 1.54 min

N-{[(2R)-1-(4-(3-Buten-1-ylamino)-6-{[(2-phenyl-1,3-thiazol-

4-yl)methyl]amino}-1,3,5-triazin-2-yl)-2-pyrrolidinyl]methyl}-

4-(methyloxy)benzenesulfonamide (19c). By following general

method B, (R)-6-(2-(aminomethyl)pyrrolidin-1-yl)-N2-(but-3-en-1-

yl)-N4-((2-phenylthiazol-4-yl)methyl)-1,3,5-triazine-2,4-diamine was

ﬁrst made, which was then treated with 4-methoxylbenzene-1-sulfonyl

chloride to aﬀord the title compound in 65% yield. LC−MS (ESI) m/z

1

(HPLC method D); H NMR (CDCl₃, 300 MHz) δ 7.60 (d, 2 H),

7.23−7.20 (d, 2 H), 4.80 (m, 1 H), 4.10 (m, 1 H), 3.50 (m, 3 H), 3.15

(m, 2 H), 2.95 (m, 1 H), 2.64−2.59 (t, 2 H), 1.95 (m, 1 H), 1.85−1.50

(m, 7 H), 1.41 (m, 2 H),1.25 (m, 6 H), 0.94−0.89 (t, 6 H). HRMS (M

+ H)⁺calcd for [C₂₄H₃₉N₇O₂S + H] 490.2959; found 490.2937.

N-{[(2S)-1-(4-(3-Buten-1-ylamino)-6-{[(2-phenyl-1,3-thiazol-

4-yl)methyl]amino}-1,3,5-triazin-2-yl)-2-pyrrolidinyl]methyl}-

4-(triﬂuoromethyl)benzenesulfonamide (16). By use of 1-(2-

phenyl-1,3-thiazol-4-yl)methanamine as NR₂, N-[(2S)-2-pyrrolidinyl-

methyl]-4-(triﬂuoromethyl)benzenesulfonamide TFA salt as NR₃, and

3-buten-1-amine as NR₁, the title compound was prepared by

following general method A. MS (ESI) m/z [M + 1]⁺= 645.3, t_R=

1

[M + 1]⁺= 607.3, t_R= 6.56 min (HPLC method B); H NMR (400

MHz, CD₃OD) δ 7.99 (d, 2H), 7.60−7.80 (m, 2H), 7.4 (m, 4H),

7.10−6.80 (dd, 2H), 5.80 (m, 1H), 5.14−5.03 (m, 2H), 4.80−4.68 (m,

2H), 4.23 (br, 2H), 3.85−3.76 (d, 3H), 3.40−3.20 (m, 2H), 2.40−2.20

(m, 2H), 2.00−1.89 (m, 4H). HRMS (M + H)⁺calcd for

[C₂₉H₃₄N₈O₃S₂+ H] 607.2268; found 607.2247.

N-{[(2R)-1-(4-(3-Buten-1-ylamino)-6-{[(2-phenyl-1,3-thiazol-

4-yl)methyl]amino}-1,3,5-triazin-2-yl)-2-pyrrolidinyl]methyl}-

benzenesulfonamide (19d). By following general method B, (R)-6-

(2-(aminomethyl)pyrrolidin-1-yl)-N2-(but-3-en-1-yl)-N4-((2-phenyl-

thiazol-4-yl)methyl)-1,3,5-triazine-2,4-diamine was ﬁrst made, which

was then treated with phenylsulfonyl chloride to aﬀord the title

compound in 23% yield. LC−MS (ESI) m/z [M + 1]⁺= 577.3, t_R=

8.74 min (HPLC method C). HRMS (M + H)⁺calcd for

[C₂₈H₃₂N₈O₂S₂+ H] 577.2162; found 577.2150.

1

7.82 min (HPLC method B). H NMR (400 MHz, CD₃OD) δ 7.99

(d, J = 8.8 Hz, 1H), 7.94−7.90 (m, 2H), 7.86 (d, J = 8.8 Hz, 1H), 7.73

(d, J = 8.4 Hz, 1H), 7.46 (m, 3H), 5.80 (m, 1H), 5.13−5.02 (m, 2H),

4.73 (m, 2H), 4.27 (m, 1H), 3.65 (m, 1H), 3.48 (m, 3H), 3.19−3.01

(m, 2H), 2.38 (m, 1H), 2.30 (m, 1H), 2.06−1.92 (m, 4H). HRMS (M

+ H)⁺calcd for [C₂₉H₃₁F₃N₈O₂S₂+ H] 645.2036; found 645.2009.

N-{[(3S)-1-(4-(3-Buten-1-ylamino)-6-{[(2-phenyl-1,3-thiazol-

4-yl)methyl]amino}-1,3,5-triazin-2-yl)-3-pyrrolidinyl]methyl}-

4-(triﬂuoromethyl)benzenesulfonamide (17). By use of 1-(2-

phenyl-1,3-thiazol-4-yl)methanamine as NR₂, (S)-N-(pyrrolidin-3-

ylmethyl)-4-(triﬂuoromethyl)benzenesulfonamide TFA salt as NR₃,

and 3-buten-1-amine as NR₁, the title compound was prepared by

following general method A. MS (ESI) m/z [M + 1]⁺= 645.3, t_R=

7.63 min (HPLC method B). ¹H NMR (400 MHz, DMSO-d₆) δ 8.08

(m, 2H), 7.99 (m, 4H), 7.91 (d, J = 2.8 Hz, 1H), 7.89 (s, 1H), 7.48 (d,

J = 5.2 Hz, 2H), 7.47 (s 1H), 5.78 (m, 1H), 5.11−4.89 (m, 2H), 4.64

(s, 2H), 3.58 (br, 2H), 3.45 (m, 5H), 3.20 (m, 1H), 2.83 (t, J = 5.2,

2H), 2.30 (m, 2H), 2.18 (m, 1H), 1.97 (m, 1H), 1.64 (m, 1H). HRMS

(M + H)⁺calcd for [C₂₉H₃₁F₃N₈O₂S₂+ H] 645.2036; found 645.2018.

HRMS (M + H)⁺calcd for [C₂₉H₃₁F₃N₈O₂S₂+ H] 645.2036; found

645.2007.

N-{[(2R)-1-(4-(3-Buten-1-ylamino)-6-{[(2-phenyl-1,3-thiazol-

4-yl)methyl]amino}-1,3,5-triazin-2-yl)-2-pyrrolidinyl]methyl}-

cyclopropanesulfonamide (19e). By following general method B,

(R)-6-(2-(aminomethyl)pyrrolidin-1-yl)-N2-(but-3-en-1-yl)-N4-((2-

phenylthiazol-4-yl)methyl)-1,3,5-triazine-2,4-diamine was ﬁrst made,

which was then treated with cyclopropanesulfonyl chloride to aﬀord

the title compound in 40% yield. LC−MS (ESI) m/z [M + 1]⁺

=

541.3, t_R= 5.71 min (HPLC method B). HRMS (M + H)⁺calcd for

[C₂₅H₃₂N₈O₂S₂+ H] 541.2162; found 541.2148.

(R)-N-((1-(4-(Butylamino)-6-(((2-(thiophen-2-yl)thiazol-4-yl)-

methyl)amino)-1,3,5-triazin-2-yl)pyrrolidin-2-yl)methyl)-3-me-

thoxybenzenesulfonamide and (R)-N-((1-(4-(Butylamino)-6-

(((2-(thiophen-2-yl)thiazol-4-yl)methyl)amino)-1,3,5-triazin-2-

yl)pyrrolidin-2-yl)methyl)-3-hydroxybenzenesulfonamide (20i-

OMe and 20i). By following general method B, (R)-6-(2-

(aminomethyl)pyrrolidin-1-yl)-N2-butyl-N4-((2-(thiophen-2-yl)-

thiazol-4-yl)methyl)-1,3,5-triazine-2,4-diamine TFA salt was ﬁrst

made. To a solution of this intermediate (0.1 g, 0.23 mmol) in

THF (10 mL) were added K₂CO₃(0.09 g, 0.68 mmol) and 3-

methoxybenzene-1-sulfonyl chloride (0.07 g, 0.34 mmol). The

N-{[(3R)-1-(4-(3-Buten-1-ylamino)-6-{[(2-phenyl-1,3-thiazol-

4-yl)methyl]amino}-1,3,5-triazin-2-yl)-3-pyrrolidinyl]methyl}-

4-(triﬂuoromethyl)benzenesulfonamide (18). By use of 1-(2-

phenyl-1,3-thiazol-4-yl)methanamine as NR₂, (R)-N-(pyrrolidin-3-

ylmethyl)-4-(triﬂuoromethyl)benzenesulfonamide TFA salt as NR₃,

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reaction mixture was stirred at 60 °C for 2 h and then concentrated in

vacuo. The residue was dissolved in EtOAc (20 mL) and washed with

H₂O (10 mL × 3). The organic phase was dried over Na₂SO₄, ﬁltered,

and concentrated in vacuo to give N-({(2R)-1-[4-(butylamino)-6-

({[2-(2-thienyl)-1,3-thiazol-4-yl]methyl}amino)-1,3,5-triazin-2-yl]-2-

pyrrolidinyl}methyl)-3-(methyloxy)benzenesulfonamide (20i-OMe)

as a crude product. The crude product was dissolved in anhydrous

CH₂Cl₂(10 mL), and the solution was cooled to 0 °C. To this

solution, BBr₃(0.17 g, 0.68 mmol) was added dropwise. The mixture

was stirred at 0 °C for 3 h and then was quenched by the addition of

ice-cold water (20 mL). Upon cooling, the mixture was extracted with

CH₂Cl₂(10 mL × 3) and the organic phase was combined, dried over

Na₂SO₄, ﬁltered, and concentrated in vacuo. The residue was puriﬁed

by preparative HPLC (method F, MeCN/H₂O = 5/3) to aﬀord the

title compound (20i) (57 mg, 42%). LC−MS (ESI) m/z [M + 1]⁺=

601.2, t_R= 1.45 min (method D); ¹H NMR (CDCl₃, 300 MHz) δ 8.2

(1 H), 7.7 (1 H), 7.0−7.6 (8 H), 5.7−6.1 (1 H), 4.5−5.5 (2 H), 4−4.5

(1H), 3.3−3.7 (6 H), 0.8−2.1 (11 H). HRMS (M + H)⁺calcd for

[C₂₆H₃₂N₈O₃S₃+ H] 601.1832; found 601.1809.

N-({(2R)-1-[4-(Butylamino)-6-({[2-(2-thienyl)-1,3-thiazol-4-yl]-

methyl}amino)-1,3,5-triazin-2-yl]-2-pyrrolidinyl}methyl)-4-hy-

droxybenzenesulfonamide (20j). The title compound was

prepared analogously to the synthesis of compound 20i with

additional preparative HPLC puriﬁcation (method F) in 40% yield.

LC−MS (ESI) m/z [M + 1]⁺= 601.2, t_R= 1.43 min (method D); ¹H

NMR (CDCl₃, 300 MHz) δ 7.8 (1 H), 6.8−7.6 (s or d, 8 H), 5.7−5.9

(1 H), 4.7 (2 H), 4.1−5.0 (3 H), 3.1−3.4 (m, 6 H), 1.8−1.9 (4 H),

1.5−1.6 (2 H), 1.3−1.4 (2 H), 0.9 (t, 3 H). HRMS (M + H)⁺calcd for

[C₂₆H₃₂N₈O₃S₃+ H] 601.1832; found 601.1812.

(methylsulfonyl)benzenesulfonamide (20e). The title compound

was prepared analogously to the synthesis of 20i-OMe with additional

preparative HPLC puriﬁcation (method F) in 47% yield. LC−MS

1

(ESI) m/z [M + 1]⁺= 663.2, t_R= 1.45 min (method D); H NMR

(CDCl₃, 300 MHz) δ 9.2 (1 H), 8.2 (d, 1 H), 7.8−8.0 (s or d, 4 H),

7.0−7.5 (s or d, 4 H), 5.6 (s, 1 H), 4.6−5.0 (d, 2 H), 4.3 (s, 1 H), 3.2−

3.6 (m, 4 H), 3.0 (s, 3 H), 2.8−3.0 (m, 2 H), 1.3−2.1 (m, 8 H), 0.8 (t,

3 H). HRMS (M + H)⁺calcd for [C₂₇H₃₄N₈O₄S₄+ H] 663.1659;

found 663.1641.

N-({(2R)-1-[4-(Butylamino)-6-({[2-(2-thienyl)-1,3-thiazol-4-yl]-

methyl}amino)-1,3,5-triazin-2-yl]-2-pyrrolidinyl}methyl)-4-cya-

nobenzenesulfonamide (20f). The title compound was prepared

analogously to the synthesis of 20i-OMe with additional preparative

HPLC puriﬁcation (method F) in 39% yield. LC−MS (ESI) m/z [M +

1]⁺= 610.2, t_R= 1.49 min (method D). ¹H NMR (CDCl₃, 300 MHz)

δ 9.0 (1 H), 8.0−8.3 (d, 1 H), 7.0−8.0 (s or d, 8 H), 6.6 (1 H), 4.7 (2

H), 4.0−4.3 (s, 1 H), 2.8−3.6 (m, 6 H), 1.8−2.5 (4 H), 1.5−1.6 (2

H), 1.3−1.4 (2 H), 0.8 (t, 3 H). HRMS (M + H)⁺calcd for

[C₂₇H₃₁N₉O₂S₃+ H] 610.1836; found 610.1814.

N-({(2R)-1-[4-(Butylamino)-6-({[2-(2-thienyl)-1,3-thiazol-4-yl]-

methyl}amino)-1,3,5-triazin-2-yl]-2-pyrrolidinyl}methyl)-3-cya-

nobenzenesulfonamide (20g). The title compound was prepared

analogously to the synthesis of 20i-OMe with additional preparative

HPLC puriﬁcation (method F) in 48% yield. LC−MS (ESI) m/z [M +

1]⁺= 610.2, t_R= 1.49 min (method D); ¹H NMR (CDCl₃, 300 MHz)

δ 8.9 (1 H), 8.3 (1 H), 7.0−8.2 (8 H), 6.0−6.5 (s, 1 H), 4.7 (2 H),

4.0−4.5 (s, 1 H), 2.8−3.6 (m, 6 H), 1.9−2.1 (4 H), 1.5−1.6 (2 H),

1.3−1.4 (2 H), 0.9 (t, 3 H). HRMS (M + H)⁺calcd for

[C₂₇H₃₂N₉O₂S₃+ H] 610.1841; found 610.1842.

N-({(2R)-1-[4-(Butylamino)-6-({[2-(2-thienyl)-1,3-thiazol-4-yl]-

methyl}amino)-1,3,5-triazin-2-yl]-2-pyrrolidinyl}methyl)-4-

chlorobenzenesulfonamide (20a). The title compound was

prepared analogously to the synthesis of compound 20i-OMe with

additional preparative HPLC puriﬁcation (method F) in 45% yield.

LC−MS (ESI) m/z [M + 1]⁺= 619.2, t_R= 1.56 min (method D); ¹H

NMR (CDCl₃, 300 MHz) δ 8.2 (1 H), 8.0 (1H), 7.0−7.8 (8 H), 5.5−

6.0 (1 H), 4.7 (2 H), 4.3 (1 H), 2.8−3.2 (6 H), 1.1−2.1 (8 H), 0.8 (3

H). HRMS (M + H)⁺calcd for [C₂₆H₃₁ClN₈O₂S₃+ H] 619.1493;

found 619.1463.

N-({(2R)-1-[4-(Butylamino)-6-({[2-(2-thienyl)-1,3-thiazol-4-yl]-

methyl}amino)-1,3,5-triazin-2-yl]-2-pyrrolidinyl}methyl)-4-ﬂu-

orobenzenesulfonamide (20h). The title compound was prepared

analogously to the synthesis of 20i-OMe with additional preparative

HPLC puriﬁcation (method F) in 41% yield. LC−MS (ESI) m/z [M +

1]⁺= 603.2, t_R= 1.51 min (method D); ¹H NMR (CDCl₃, 300 MHz)

δ 9.1 (1 H), 8.1−8.3 (1 H), 7.0−8.0 (8 H), 6.1−6.3 (1 H), 4.7 (2 H),

4.1−4.4 (1 H), 2.8−3.7 (m, 6 H), 1.8−2.0 (3 H), 1.3−1.5 (3 H), 1.2−

1.4 (3 H), 0.85 (3 H). HRMS (M + H)⁺calcd for [C₂₆H₃₁FN₈O₂S₃+

H] 603.1789; found 603.1779.

N-({(2R)-1-[4-(Butylamino)-6-({[2-(2-thienyl)-1,3-thiazol-4-yl]-

methyl}amino)-1,3,5-triazin-2-yl]-2-pyrrolidinyl}methyl)-3-

chlorobenzenesulfonamide (20b). The title compound was

prepared analogously to the synthesis of 20i-OMe with additional

preparative HPLC puriﬁcation (method F) in 48% yield. LC−MS

Aﬃnity Selection. Selections were done using streptavidin matrix

tips (Phynexus) and chemically biotinylated ADAMTS-5 (262−624)

protein. Three rounds of selections were performed. Each tip in each

round of selection had 10 μg of protein loaded onto it. Tips were

prepared as follows: tips were washed three times with blocking buﬀer

(5% casein in 1× selection buﬀer (50 mM Tris (pH 7.5), 150 mM

NaCl, 0.05% Brij35, 2 mM β-mercaptoethanol (BME), and 1.0 mg/

mL sheared salmon sperm DNA (sssDNA, Ambion))) and stored for

12 h at 4 °C in the same buﬀer. Tips were rinsed two times with

blocking buﬀer. A ﬁnal buﬀer exchange was performed with 1.0 mg/

mL sssDNA in 1× selection buﬀer, and the tips were stored in 80 mL

of the sssDNA in 1× selection buﬀer for 12 h at 4 °C.

For selection round 1, 10 mg of ADAMTS-5 protein was

immobilized on a previously prepared streptavidin matrix tip and the

tip was washed four times with selection buﬀer. An amount of 5 nmol

of DEL-A was diluted in 60 mL of 1× selection buﬀer and passed over

the streptavidin matrix tip for 1 h at room temperature. The tip was

washed 8 times with 1× selection buﬀer and two times with DNA free

1× selection buﬀer. Binders were eluted by passing heated (80 °C)

DNA free 1× selection buﬀer over it for 12 min. The eluted material

was passed over a fresh streptavidin matrix tip for 10 min at room

temperature to remove any denatured protein (postclear). The

postclear step was repeated a second time to the round 1 selection

output (1 μL of the round 1 selection output was retained to be used

for qPCR in order to monitor the output from this round of selection).

The remaining round 1 selection output was brought to 60 μL by

adding the necessary volume of sssDNA and selection buﬀer.

For selection round 2, the above selection procedure was repeated

with fresh protein, a previously prepared fresh tip (washed an

additional four times with selection buﬀer before use), and the round 1

selection output. At the end of round 2, the selection output was

1

(ESI) m/z [M + 1]⁺= 619.2, t_R= 1.54 min (method D); H NMR

(CDCl₃, 300 MHz) δ 9.0 (1 H), 8.0−8.3 (d, 1 H), 7.0−7.6 (s or d, 8

H), 6.1 (s, 1 H), 4.7 (2 H), 4.3 (s, 1 H), 2.8−3.6 (m, 6 H), 1.9−2.0 (4

H), 1.5−1.6 (2 H), 1.3−1.4 (2 H), 0.9 (t, 3 H). HRMS (M + H)⁺

calcd for [C₂₆H₃₁ClN₈O₂S₃+ H] 619.1493; found 619.1465.

N-({(2R)-1-[4-(Butylamino)-6-({[2-(2-thienyl)-1,3-thiazol-4-yl]-

methyl}amino)-1,3,5-triazin-2-yl]-2-pyrrolidinyl}methyl)-3,4-di-

methylbenzenesulfonamide (20c). The title compound was

prepared analogously to the synthesis of 20i-OMe with additional

preparative HPLC puriﬁcation (method F) in 46% yield. LC−MS

1

(ESI) m/z [M + 1]⁺= 613.2, t_R= 1.55 min (method D); H NMR

(CDCl₃, 300 MHz) δ 9.4 (1 H), 8.2 (1 H), 7.0−7.6 (5 H), 5.8 (s, 1

H), 4.7 (2 H), 4.0−4.4 (s, 1 H), 2.8−3.6 (m, 6 H), 2.3 (6 H),1.9−2.0

(4 H), 1.5−1.6 (2 H), 1.3−1.4 (2 H), 0.9 (t, 3 H). HRMS (M + H)⁺

calcd for [C₂₈H₃₆N₈O₂S₃+ H] 613.2196; found 613.2186.

N-({(2R)-1-[4-(Butylamino)-6-({[2-(2-thienyl)-1,3-thiazol-4-yl]-

methyl}amino)-1,3,5-triazin-2-yl]-2-pyrrolidinyl}methyl)-4-bi-

phenylsulfonamide (20d). The title compound was prepared

analogously to the synthesis of 20i-OMe with additional preparative

HPLC puriﬁcation (method F) in 37% yield. LC−MS (ESI) m/z [M +

1]⁺= 661.2, t_R= 1.62 min (method D); ¹H NMR (CDCl₃, 300 MHz)

δ 9.1 (s, 1 H), 8.1−8.3 (d, 1 H), 7.0−8.0 (s or d, 13 H), 6.0 (d, 1 H),

4.7 (m, 2 H), 4.1−4.3 (m, 1 H), 2.8−3.6 (m, 6 H), 1.8−2.6 (t, 4 H),

1.4−1.6 (m, 3 H), 1.2−1.4 (m, 3 H), 0.9 (t, 3 H). HRMS (M + H)⁺

calcd for [C₃₂H₃₆N₈O₂S₃+ H] 661.2196; found 661.2169.

N-({(2R)-1-[4-(Butylamino)-6-({[2-(2-thienyl)-1,3-thiazol-4-yl]-

methyl}-amino)-1,3,5-triazin-2-yl]-2-pyrrolidinyl}methyl)-4-

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postcleared two times as previously described (5 μL of the round 2

selection output was retained to be used for qPCR in order to monitor

the output from this round of selection). The remaining round 2

selection output was brought to 60 μL by adding the necessary volume

of sssDNA and selection buﬀer.

For selection round 3, the selection procedure was repeated with

fresh protein, a previously prepared fresh tip (washed an additional

four times with selection buﬀer before use), and the round 2 selection

output. The postclear steps were not repeated at the end of round 3.

Quantitative PCR was run with the outputs from each round of

selection to assess selection yields for each step. The round 3 output

was sequenced using Roche/454 technology (454 contract sequenc-

ing). The no target control selection was performed in the same

fashion but without the protein input.

ADAMTS-5 Assay. The biological activity of the ADAMTS-5

compounds was measured using an assay described by Zhang et al.²²

WAAG-3R substrate was purchased from Anaspec (catalog no. 60431-

1). Truncated ADAMTS-5 (262−624) was expressed with a C-term

aﬃnity tag in the Chinese hamster ovary cell line, puriﬁed from

conditioned medium via aﬃnity tag, released from matrix by

proteolytic cleavage, and further resolved by size exclusion

chromatography. Enzyme and substrate concentrations were 60 nM

and 25 μM, respectively. The 1× buﬀer consists of 50 mM HEPES, pH

7.5, 100 mM NaCl, 5 mM CaCl₂, 0.1% CHAPS, and 5% glycerol.

Assay was run in 384-well black plates (Greiner catalog no. 781209).

Compounds were suspended in 100% DMSO at 10 mM and diluted

serially (ﬁnal compound concentration 1% DMSO and 1× assay

buﬀer) and preincubated with the enzyme for 15 min. Substrate was

added, and the plate was read at every 30 s for 1 h at 37 °C. Excitation

and emission wavelengths were 340 and 420 nm, respectively. The

enzyme rate in the presence of inhibitor was compared to the rate with

1% DMSO alone to determine percent inhibition at a particular

inhibitor concentration. Compound IC₅₀values were determined using

a sigmoidal dose−response curve ﬁtting routine (Graphpad Prism).

ADAMTS-4 Assay. The biological activity of the ADAMTS-4

compounds was measured using an assay described by Zhang et al.²²

ADAMTS-4, similar to that previously described in Wayne et al.,³²and

substrate concentrations were 48 nM and 25 μM respectively. The 1×

buﬀer consists of 50 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM

CaCl₂, 0.1% CHAPS, and 5% glycerol. Assay was run in 384-well black

plates (Greiner catalog no. 781209). Compounds were suspended in

100% DMSO at 10 mM and diluted serially (ﬁnal compound

concentration 1% DMSO and 1× assay buﬀer) and preincubated with

the enzyme for 15 min. The substrate (WAAG-3R) was added, and the

plate was read every 30 s for 1 h at 37 °C. Excitation and emission

wavelengths were 340 and 420 nm, correspondingly. IC₅₀values were

determined as described above.

TACE Assay. The assay was run with TACE enzyme (R&D 930-

ADB) and TACE substrate (R&D ES003). The assay was run in 1×

TACE buﬀer: 25 mM Tris (pH 8.0), 2.5 μM ZnCl₂, and 0.1% Brij-35.

TACE enzyme and substrate concentrations were 20 nM and 100 μM,

respectively. Compounds were diluted serially and preincubated with

the enzyme for 15 min. Substrate was added, and the plate was read

every 30 s for 30 min. Excitation and emission wavelengths were 355

and 405 nm, correspondingly. IC₅₀values were determined as

described above.

MMP-13 Assay. MMP-13 enzyme was purchased from R&D

Systems (R&D 511-MM). The assay was run in 50 mM HEPES (pH

7.5), 150 mM NaCl, 10 mM CaCl₂, 1 μM ZnAc₂, and 600 μM

CHAPS. Substrate was custom synthesized 5-FAM-TPGPLGL[Dap-

(DNP)]ARRK(5-TAMRA)-amide. Compounds were diluted serially

in DMSO (ﬁnal 1%) and added to the plate (Greiner catalog no.

784076). Assay buﬀer, substrate (ﬁnal 1 μM), and MMP-13 enzyme

(ﬁnal 480 pM) were added sequentially to the plate and incubated at

room temperature for 45 min. The reaction was stopped by adding

EDTA (ﬁnal 10 mM). The assay plates were read at 485 nm

excitation/530 nm emission with a 505 nm dichroic ﬁlter. IC₅₀values

were determined as described above.

7.5), 150 mM NaCl, 1 μM ZnAc, 10 mM CaCl₂, and 600 μM CHAPS.

Substrate FAM (5-carbosyﬂuorescein)-AE*LQGRPISIAK-

TAMRA(5(6)-carbosytetramethylrhodamine) was custom synthesized

by Enzyme Systems Products, Inc. Compounds were diluted serially in

DMSO (ﬁnal 1%) and added to the plate (Greiner no. 784076). Assay

buﬀer, substrate (ﬁnal 2.5 μM), and ADAMTS-1 enzyme (ﬁnal 20

nM) were added to the plates and incubated at room temperature for

2 h. The reaction was stopped by adding EDTA (ﬁnal 10 mM). The

assay plates were read at 485 nm excitation and 530 nm emission with

a 505 nm dichroic ﬁlter. IC₅₀values were determined as described

above.

Rat PK Study. Characterization of the pharmacokinetics of 15f,

13g, and 13e (Table 6) was done after cassette administration of single

intravenous (iv) and oral (po) doses in male Sprague−Dawley rats.

The iv infusion dose was administered to fed rats through the femoral

vein at 4 mL/kg over 30 min (0.8−0.9 mg compound/kg).

Formulation was a ﬁltered solution in 5% DMSO and 20% HPbCD

(hydroxypropyl-β-cyclodextrin, also known as Cavitron) in water, pH

3−5, and sampling was done at 0, 5, 15, 30, 32, 35, 45, 60, 90, 120,

180, 240, 360, 480, 600, and 1440 min. The po dose was administered

by gavage at 16 mL/kg (2.0 mg of 15f/kg, 0.5 mg of 13g/kg, and 0.6

mg of 13e/kg) to fasted rats (fed 4 h postdose). Formulation was a

ﬁltered solution in 5% DMSO and 6% HPbCD in water, pH 3−5, and

sampling was done at 0, 5, 15, 30, 45, 60, 90, 120, 180, 240, 360, 480,

600, and 1440 min. Blood was collected from the jugular vein, and

plasma was analyzed by LC/MS/MS.

Human OA Cartilage Explant Assay. Tissue was obtained from

a 68 year old human osteoarthritis patient within 24 h of undergoing

total knee replacement (NDRI, Philadelphia, PA) under IRB approval

and in compliance with UK Human Tissue Act 2004 guidelines.

Cartilage tissue was processed from the bone into uniform 3 mm

diameter disks using a scalpel and leather punch in a sterile hood.

Disks were randomized and placed into 96-well tissue culture plates in

DMEM supplemented with 0.5% FBS, Penn/Strep and ^L-glutamine.

Following a 3-day equilibration to culture, the medium was removed

and fresh medium containing compound and control treatments and

recombinant human IL-1β and oncostatin M was added to the wells in

replicates of seven per treatment group. IL-1β and oncostatin M were

added to the wells in a stepwise fashion (0.5 ng/mL each on days 0−7,

5.0 ng/mL each on days 7−14, and 50 ng/mL each on days 14−18).

Each change of cytokine levels required a complete medium change,

and fresh medium including treatments at consistent concentration

was made (days 7 and 14). Conditioned medium time points were

sampled throughout the study and tested for ³⁷⁴ARGS neoepitope and

GAG release.

³⁷⁴ARGS Neoepitope Release Detection. Levels of ³⁷⁴ARGS

were quantiﬁed using a sandwich ELISA format as described

previously.²⁹Brieﬂy, assay plates (Perkin-Elmer) were coated [10

μg/mL] overnight at 4 °C with a monoclonal antihuman aggrecan

capture antibody speciﬁc to the keratin sulfate domains (Invitrogen,

AHP0012). Plates were washed in 1× DELFIA wash buﬀer (Perkin-

Elmer) and an equal volume (25 μL) of 1× DELFIA assay buﬀer and

cartilage explant samples were added to the wells according to the

experimental plate layout. Plates were incubated for 2 h at 37 °C,

washed, and incubated with a biotinylated ³⁷⁴ARGS neoepitope

speciﬁc detection antibody (OA-1, 50 μL/well at 1ug/mL) for 1 h at

room temperature and washed. Released ³⁷⁴ARGS neoepitope was

detected using streptavidin−europium (50 μL of 100 ng/mL for 30

min at room temperature) and DELFIA enhancement solution (200

μL/well for 3−5 min on a shaker) followed by europium signal

acquisition (320 nm excitation, 615 nm emission) on an Envision plate

reader.

Glycosaminoglycan Release Detection. Release of glycosami-

noglycan (GAG) from cartilage was quantiﬁed in the tissue culture

medium using the 1,9-dimethylmethylene Blue (DMMB) dye binding

procedure as described previously.²⁸

ADAMTS-1 Assay. ADAMTS-1 was purchased from R&D

Systems (no. 2197-AD). The assay was run in 50 mM HEPES (pH

7077

dx.doi.org/10.1021/jm300449x | J. Med. Chem. 2012, 55, 7061−7079

Journal of Medicinal Chemistry

Article

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S. L.; Benjamin, D. R.; Carlson, N. R.; Centrella, P. A.; Chiu, C. H.;

Creaser, S. P.; Cuozzo, J. W.; Davie, C. P.; Ding, Y.; Franklin, G. J.;

Franzen, K. D.; Gefter, M. L.; Hale, S. P.; Hansen, N. JV.; Israel, D. I.;

Jiang, J.; Kavarana, M. J.; Kelley, M. S.; Kollmann, C. S.; Li, F.; Lind,

K.; Mataruse, S.; Medeiros, P. F.; Messer, J. A.; Myers, P.; O’Keefe, H.;

Oliff, M. C.; Rise, C. E.; Satz, A. L.; Skinner, S. R.; Svendsen, J. L.;

Tang, L.; Vloten, K. V.; Wagner, R. W.; Yao, G.; Zhao, B.; Morgan, B.

A. Design, Synthesis and Selection of DNA-Encoded Small-Molecule

Libraries. Nat. Chem. Biol. 2009, 5, 647−654.

(18) Graybill, T. L.; King, B. W.; Fosbenner, D. T.; Keller, P. M.;

Morgan, B. A.; Clark, M. A.; Cuozzo, J. DNA-Encoded Libraries: A

New Resource for Innovative Hit Identiﬁcation. Abstract of Papers,

237th National Meeting of the American Chemical Society, Salt Lake

City, UT, U.S., March 22−26, 2009; American Chemical Society:

Washington, DC, 2009; MEDI-297.

ASSOCIATED CONTENT

* Supporting Information

■

S

Experimental details for the synthesis of the DNA encoded

library, the ADAMTS-5 inhibition experiment with various

concentrations of ZnCl₂, the substrate−inhibitor competition

experiment, the solubility and artiﬁcial membrane permeability

assays, and rat liver microsomal stability assay. This material is

available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*For H.D.: phone, 1-781-795-4302; fax, 1-781-795-4496; e-

mail, hongfeng.x.deng@gsk.com. For C.C.A.-M.: phone, 1-781-

795-4272; fax, 1-781-795-4496; e-mail, christopher.c.arico-

muendel@gsk.com.

■

Present Addresses

^∞EnVivo Pharmaceuticals, 500 Arsenal Street, Watertown, MA

02472, U.S.

^×X-Chem Inc., 100 Beaver Street, Waltham, MA 02453, U.S.

^●Biogen Idec, 14 Cambridge Center, Cambridge, MA 02142,

U.S.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We acknowledge ChemPartner Ltd (Shanghai, China) for

preparing compounds 15 and 20; Jayshree Mistry in PTS,

GlaxoSmithKline, for managing this contract synthesis eﬀort;

and Dr. Christine Donahue, PTS, GlaxoSmithKline, for her

suggestions regarding the enzyme mechanism.

ABBREVIATIONS USED

■

ELT, encoded library technology; OA, osteoarthritis; siRNA,

small interfering RNA; DEL, DNA encoded library; AOP, 15-

amino-4,7,10,13-tetraoxapentadecanoic acid; BB, building

block; Nvoc, o-nitroveratryloxycarbonyl; NTC, no-target

control; TACE, tumor necrosis factor α-converting enzyme;

MMP, matrix metalloproteinase; GAG, glycosaminoglycan; IL-

1β, interleukin-1β; OSM, oncostatin M; PK, pharmacokinetic;

DIPEA, diisopropylethylamine

ADDITIONAL NOTE

■

The human biological samples were sourced ethically, and their

research use was in accord with the terms of the informed

consents. All studies were conducted after review by the GSK

Institutional Animal Care and Use Committee and in

accordance with the GSK Policy on the Care, Welfare and

Treatment of Laboratory Animals.

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dx.doi.org/10.1021/jm300449x | J. Med. Chem. 2012, 55, 7061−7079

Article Doi

Discovery of highly potent and selective small molecule ADAMTS-5 inhibitors that inhibit human cartilage degradation via encoded library technology (ELT)

DOI: 10.1021/jm300449x

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