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Metastatic Melanoma: Insights Into the Evolution of the

Treatments and Future Challenges
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DOI: 10.1002/med.21404
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Metastatic Melanoma: Insights Into
the Evolution of the Treatments
and Future Challenges
Antoine Millet,1 Anthony R. Martin,1 # Cyril Ronco,1 # Stéphane Rocchi,2,3,4*

and Rachid Benhida1*
1 Institut de Chimie de Nice UMR UNS-CNRS 7272, Nice, France
2 INSERM, U1065, Centre Méditerranéen de Médecine Moléculaire (C3M), Equipe Biologie et Pathologie des
cellules mélanocytaires: de la pigmentation cutanée au mélanome, Nice, France
3 Université de Nice Sophia Antipolis, UFR de Médecine, Nice, France
4 Service de Dermatologie, Hôpital Archet II, CHU Nice, France
Published online in Wiley Online Library (wileyonlinelibrary.com).

DOI 10.1002/med.21404
䉲
Abstract: Melanoma is the deadliest form of skin cancer. While associated survival prognosis is good
when diagnosed early, it dramatically drops when melanoma progresses into its metastatic form. Prior to
2011, the favored therapies include interleukin-2 and chemotherapies, regardless of their low efficiency
and their toxicity. Following key biological findings, two new types of therapy have been approved. First,
there are the targeted therapies, which rely on small molecule B-Raf and MEK inhibitors and allow the
treatment of patients with B-Raf mutated melanoma. Second, there are the immunotherapies, with anti-
CTLA-4 and anti-PD-1 antibodies that are used for patients harboring a B-Raf wild-type status. Both
approaches have significantly improved patient survival, compared with alkylating agents, in the treatment
of unresectable melanoma. Herein, we review the evolution of the treatment of melanoma starting from
early discoveries to current therapies. A focus will be provided on drug discovery, synthesis, and mode
of action of relevant drugs and the future directions of the domain to overcome the emergence of the
resistance events.
C 2016 Wiley Periodicals, Inc. Med. Res. Rev., 37, No. 1, 98–148, 2017
Key words: melanoma; targeted therapies; B-Raf/MEK inhibitors; immunotherapies; drug resistance
1. INTRODUCTION
Among all kind of cancers, skin cancers occur frequently.1 They are divided into two cate-
gories; nonmelanoma and melanoma skin cancers. The nonmelanoma skin cancers encompass
the basal-cell and squamous-cell carcinomas together with a few other rare types of skin
# These authors contributed equally to this work.

∗ Correspondence to: Stéphane Rocchi, INSERM U1065, Team 1, Centre Méditerranéen de Médecine Moléculaire
(C3M), Bâtiment ARCHIMED, 151 Route de Saint Antoine de Ginestière, 06204 Nice Cedex 3, France; Rachid
Benhida, Institut de Chimie de Nice UMR UNS-CNRS 7272, Université Nice Sophia Antipolis, Parc Valrose, 06108
Nice Cedex 2, France. E-mail: srocchi@unice.fr, benhida@unice.fr
Medicinal Research Reviews, 37, No. 1, 98–148, 2017

C 2016 Wiley Periodicals, Inc.
METASTATIC MELANOMA r 99
cancer. Altogether, they are the most common form of cancer in many countries worldwide2
but seldom lead to death. Conversely, melanoma skin cancers represent fewer cases (less than
5% of all skin cancer cases),3 but they account for 60–80% of deaths.4 In developed countries,
skin melanoma appears with a respective incidence and mortality of 10.2 and 2.0 per 100,000
persons in the male population while lower incidence and mortality (9.3 and 1.2 per 100,000
people) in the female population is observed. Strikingly, the Caucasian population is more
affected by melanoma as its occurrence reaches up to 60 cases per 100,000 persons in 2013
in New Zealand and Australia.5 This disease is classified into four clinical subtypes according
to tumor localization and appearance: nodular melanoma, lentigo melanoma, acral lentigi-
nous melanoma, and finally the most frequent one, the superficial spreading melanoma that
accounts for 50–70% of melanoma cases.3, 6 Generally, the nodular and the acral lentiginous
melanomas present poor prognosis due to their rapid spread and progression. In the early stages
of melanoma (stages I and II), the tumor is highly localized and can be treated by surgery and
adjuvant therapy. In this case, the prognosis is often positive and when the tumor thickness
is below 2 mm, the associated 20-year survival rate reaches 75% (90% when tumor thickness
< 1 mm).7, 8 In sharp contrast, once it reaches its metastatic form (stage III local metastasis
and stage IV distant metastasis), it becomes hard to treat and the survival rate dramatically
decreases.9 The 5-year survival rate associated with the metastatic form of melanoma varies
from 5 to 19%.4, 6, 8, 10 There are notable differences in the patient survival rate depending on
the localization of metastasis in the body. For instance, when cutaneous, the 5-year survival
rate reaches 19% whereas it drops to 7–10% when the metastases are localized in the lung or
on other organs with a median survival of 8–9 months.
Melanoma originates from the malignant mutations of melanocytes. The latter are a type
of cell predominantly localized in the skin, hair follicles, and eyes where they produce melanin,
the pigment responsible for the skin and hair coloration. In the skin, melanocytes are localized
in the basal layer of epidermis, at the interface with the dermis.4, 6, 9 Melanocytes are crucial
in the defense of our body against radiations from the sun. Following UV exposure, they pro-
duce melanin through a keratinocyte-mediated signaling cascade, hence activating the tanning
response. This enables the skin to prevent UV damage and mutagenic effects. However, several
mutations in the growth regulatory genes together with the loss of some adhesion receptors
result in the proliferation and spreading of melanocytes, the growth of which is no longer
dependent on the keratinocyte-mediated signaling cascade.
The understanding of the signaling cascades involved in the deregulation of the cell growth
in melanoma has been crucial for the development of efficient therapies. Several mutations
are responsible for the occurrence and progression of melanoma. Recently, the cancer atlas
network proposed a genomic classification of melanoma. It classified the cutaneous melanoma
into four subtypes: the B-Raf, the Ras, the NF1, and finally the triple wild-type subtypes.11
The first three subtypes were all designed according to the discovery of the involvement of the
mitogen-activated protein kinase (MAPK) pathway in the melanoma apparition and spread in
2002.12 This represented a major breakthrough in the comprehension of this disease. Notably,
the discovery of the implication of a mutated form or B-Raf was crucial for the development of
new therapies. Extended studies focusing on the MAPK/ERK pathway identified the presence
of B-rapidly accelerated fibrosacroma (B-Raf) kinase mutation in about 60% of all melanoma
cases.13–17 Rat sarcoma (Ras) is also frequently mutated in melanoma (up to 10–20%) and to
a lesser extent, a mutated form of mitogen-activated protein kinase kinase (MEK) accounts
for 6–7% of cases. These mutations, located at different levels of the MAPK/ERK pathway,
are accountable for its constitutive activation and in fine cell proliferation. The MAPK/ERK
pathway has been extensively studied and remains the most frequently drug-targeted pathway
in melanoma (Fig. 1).
Medicinal Research Reviews DOI 10.1002/med

100 r MILLET ET AL.
Figure 1. Chronology of the therapies approved to treat the metastatic melanoma.
Apart from the MAPK pathway, the PI3K/Akt pathway also plays a crucial role in the
development of melanoma. This pathway regulates fundamental functions such as cell prolifer-
ation, growth, and survival.18 Initially, the activation of PI3K protein was found to pass mostly
through its direct interaction with the receptor tyrosine kinases (RTKs) and G-protein coupled
receptors (GPCRs). It was recently demonstrated that PI3K can also be activated through
N-Ras action.19, 20 It positively regulates PI3K by the direct activation of its p110β subunit,
resulting in PI3K activation. Next, PI3K allows the conversion of phosphatidylinositol-4,5-
disphosphate (PiP2 ) into phosphatidylinositol-3,4,5-triphosphate (PiP3 ) that activates protein
kinase B (Akt), and finally the transcription of proteins involved in the cell growth, and
survival.13, 19 The inactivation of the phosphatase and tensin homolog (PTEN),21 a tumor
repressor gene that is supposed to inactivate PI3K, enhances the activity of the PI3K/Akt
pathway. Hence, this pathway is frequently overexpressed within melanoma.20 Other pathways
are involved to a lesser extent in melanoma progression; for example, NF-κB, JNK/C-Jun, or
WNT pathway. Nevertheless, as there is no FDA-approved drug targeting these pathways, they
will not be reviewed herein.
Until 2011, the privileged strategy to treat metastatic melanoma was the use of nonspecific
alkylating and/or antineoplastic agents. Herein, we will review the evolution of the treatment
of melanoma; the synthetic routes to the drugs, their mode of action and related efficacy will
also be disclosed. As 2011 represents a turning point in the history of melanoma treatment, we
will divide this review into two parts.
First, we will focus on the early antimelanoma therapies (alkylating agents) prior to 2011.
In the second part, we will discuss the emergence of targeted therapies and immunotherapies
and their applications and limitations. Finally, we will underline the major challenges, the most
promising strategies to overcome them and the main perspectives toward future directions.
2. THE USE OF FIRST-GENERATION THERAPIES
The first generation of anticancer agents are cytotoxic or cytostatic compounds that inhibit
the development of tumors by affecting DNA synthesis, replication, or through other modi
operandi. Alkylating agents belong to this family and act through DNA-base alkylation. These
DNA damages, when unrepaired lead to cell cycle arrest and, subsequently, cell death.
A. Hydroxyurea, 1967
In 1967, the FDA approved hydroxyurea (HU) as the first treatment to combat malignant
melanoma. While hydroxyurea had been known for a long time and was first synthesized in
1869 by Dresler and Stein,22 its antineoplastic properties were only demonstrated a century
later.23

Scheme 1. Synthetic pathways for the DTIC (2) and temozolomide (6).
1. Mode of Action
The anticancer activity of HU may be attributed to different pathways but the predominant
one is likely ribonucleotide reductase (RNR) inhibition, by preventing conversion of ribonu-
cleotides to deoxyribonucleotides. RNR inhibition starves the DNA polymerase from de-
oxyribonucleotides at the replication forks. Without such fundamental components, the DNA
synthesis is stopped, hence leading to cell cycle arrest in the G1 /S phase.24, 25 HU demonstrated
an approximately 20% response rate (RR) in a small clinical trial combined with radiotherapy
(3 of 14 patients)23 . When administered orally, up to 60% of the dose undergoes metabolization
following an unknown mechanism.26, 27 Together with its anticancer effect, HU also exhibits
adverse effects such as bone marrow depression, gastrointestinal symptoms, and dermatological
reactions.
B. Dacarbazine, 1975
Dacarbazine (DTIC for 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxyamide, 2) was discov-
ered in 1962 by Schealy28 and first reported for the treatment of cancer a year later.29 In 1975,
it was approved by the FDA for the treatment of malignant melanoma and other diseases such
as Hodgkin lymphoma, sarcoma, or pancreatic cancer. DTIC belongs to the triazenoimida-
zole family and can be easily prepared following a two-step strategy starting from 5-amino-4-
imidazolecarboxamide (Scheme 1). First, the 5-amino group is converted into the corresponding
diazonium salt using sodium nitrite (1). Then, nucleophilic addition of dimethylamine at the
diazonium moiety affords DTIC (2), which bears the typical dimethyltriazene motif.
1. Mode of Action
The mode of action of DTIC has been extensively studied over the past three decades.30–33
It has been shown that the dimethyltriazene group is essential for DTIC anticancer activ-
ity. When administered, the compound undergoes an α-hydroxylation of one of its methyl
radicals on the dimethyltriazene group, mediated by CP-450 in the liver,33 to give the re-
sulting 5-(3-hydroxymethyl-3-methyl)imidazole-4-carboxamide (3, Scheme 2). This unstable
species rapidly eliminates formaldehyde (4) and finally forms 5-aminoimidazole-4-carboxamide
(AIC, 5) through the release of diazomethane.30 Diazomethane is the actual alkylating agent

102 r MILLET ET AL.
Scheme 2. Mode of action of the DTIC (2) and TMZ (6).
responsible for the activity of DTIC. Its highly reactive character prompts it to react with
DNA nucleobases to form mainly N7 -methylguanosine, and O6 -methylguanosine at lower
doses.30, 33, 34 These alkylated residues play a significant role in the cytotoxicity of DTIC. More
precisely, during DNA replication, O6 -methylguanosine is recognized as an adenosine substrate
by the DNA polymerase hence resulting in G:C to A:T mutational transition.34
2. Clinical Efficacy
Clinical studies initiated in 1972 disclosed an initial RR of 20.6% (88 of 426 patients).35 However,
further studies reported a 5% response rate in 201136 (12 of 220 patients) and 6% response rate
in 201337 (4 of 63 patients). In addition to the limited response rate, the use of DTIC failed
to demonstrate an increase in the overall survival compared with placebo. Due to chemical
instability in acidic media, intravenous administration was preferred over a per os route. The
major metabolite recovered in patients’ urine, AIC (5), is inactive and nontoxic.38 Similarly to
the vast majority of alkylating agents, DTIC treatment gives rise to adverse effects, the most
frequent being anorexia, nausea, and vomiting. Despite its low efficiency and response rate,
DTIC is still a milestone in the treatment of metastatic melanoma.

Figure 2. Developed structure of the fotemustine (7).
C. Temozolomide
Temozolomide (TMZ, 6) was inspired by the rise of the triazene-containing bioactive molecules
in the late 70s. It was first synthesized in 198339 using the reaction between 1 and methyliso-
cyanate (Scheme 1). TMZ was not officially approved for the treatment of metastatic melanoma
because no clear clinical benefits were disclosed over DTIC. So far, temozolomide is sometimes
used as an alternative to DTIC due to advantageous pharmacokinetic parameters (t1/2 114 min
vs. 41 min for DTIC, enhanced stability in acidic media) and possible oral administration.3, 5, 40
Indeed, unlike DTIC, TMZ is relatively stable throughout the gastrointestinal tract.41
1. Mode of Action
TMZ acts similarly to DTIC and furnishes the same mono-methyltriazine intermediate (4).
The only difference lies in the formation of this derivative. Actually, while DTIC must un-
dergo CP-450 metabolization, TMZ yields 4 via a simple hydrolysis (Scheme 2).42 Moreover,
DNA methylation using TMZ provides similar proportions of N7 -methylguanosine and O6 -
methylguanosine compared with DTIC. Clinical trials concerning TMZ will not be discussed
since the results of phase III did not show any improved activity compared to DTIC in the
treatment of metastatic melanoma,43 resulting in its failure to be accepted by the FDA. Several
side effects have been associated with TMZ treatment, including thrombocytopenia, nausea,
vomiting, anorexia, and gastrointestinal troubles as the most frequent.
D. Fotemustine
Fotemustine (7) belongs to the class of nitrosourea anticancer agents and acts through DNA-
alkylation (Fig. 2).44 It leads to the formation of O6 - and N7 -chloroethylguanosine or, if
the chloroethyl is hydrolyzed, O6 - and N7 -hydroxyethylguanosine derivatives. These alkylated
residues exert their activity by means of a G-C interstrand crosslink or DNA single strand
break.45, 46
Fotemustine has been evaluated in a phase III clinical trial and compared with DTIC.47
The study failed to demonstrate significant therapeutic improvement when using fotemustine.
Nonetheless, this trial highlighted the fairly improved activity of fotemustine compared with
DTIC for the treatment of patients with brain metastases. Thus, it is approved for the treatment
of metastatic melanoma, but its use is restricted to Europe.
E. Interleukin-2, 1998
Following therapies based on DNA-damaging agents, a new therapeutic option emerged in the
late 1990s. It consisted of boosting the immune system to help the body fight against several
diseases, including HIV and cancer.48 The embodiment of this approach was the discovery and
the study of the cytokines family, a broad category of small proteins (5–20 kDa) involved in the
maturation, growth, and response of various immune cells.

104 r MILLET ET AL.
Figure 3. Schematic overview of the MAPK/ERK (blue) and the PI3K/Akt (pink) signaling pathways. Approved
therapies for the treatment of metastatic melanoma are indicated in green.
Interleukin-2 (IL-2) is a 15.5-kDa protein that was extensively studied from its discovery
in the 1970s and was one of the first cytokines to be characterized.49, 50 Its gene was cloned in
1984 and its structure was solved in 1992.51 The study of its mode of action revealed that IL-2
is recruited and bound to a receptor composed of three subunits: IL-2Rα, IL-2Rβ, and γ c . The
assembly of this three-membered receptor triggers the proliferation of T (CD4+ and CD8+ ), B,
and NK cells, thus making IL-2 crucial for immune homeostasis.48, 52 However, IL-2 displays
relatively low efficiency with about 10% response rate53–55 and is associated with severe side
effects including inflammatory response syndrome, hypotension, nausea, vomiting, diarrhea,
and so on.50, 53 Nonetheless, the FDA approved the use of IL-2 in the treatment of malignant
melanoma in 1998.
3. IMPLICATION OF MUTATED MAPK/ERK PATHWAY IN MELANOMA
A. MAPK/ERK Pathway Signaling

As mentioned before, the MAPK/ERK pathway plays a pivotal role in the mutagenesis and
proliferation of melanoma. Under normal circumstances, the wild-type MAPK/ERK pathway
is stimulated by extracellular stimuli such as growth factors or cytokines (Fig. 3).56 The bind-
ing of such mediators to the RTKs (EGFR, IGF1, or PDF) induces their dimerization and
autophosphorylation at the C-terminus region. Then, they recruit several proteins that drive
the activation of Ras by a GDP to GTP ligand switch.
As a consequence, the GTP-bound form of Ras recruits Raf kinase isoforms (A, B, and C)
to the plasma membrane where they are dimerized and activated by phosphorylation.57 In their
active form, Raf isoforms act as MAP kinase kinase (MAPKK) and activate MEK1 and MEK2,

which then catalyze the activation of ERK1 and ERK2. Then, ERK1/2 phosphorylates a large
variety of nuclear and cytoplasmic substrates involved in cell proliferation, differentiation, and
survival.58 Nonetheless, frequent mutations along this pathway affect Ras, Raf, and/or MEK
and result in its constitutive activation in cancer cells.
B. Ras
Ras, the upstream member of the MAPK/ERK pathway, is a GTPase expressed as three closely
related isoforms; H-Ras, K-Ras, and N-Ras. The latter is highly predominant in melanoma and
promotes oncogenesis when mutated. Under normal conditions, the deactivation rate of N-Ras
is highly dependent on the GTP-hydrolysis rate. It is assisted by both the glutamine in position
61 (Q61) and the GTPase-activating protein (GAP). Hence, residue Q61 and GAP are necessary
to maintain Ras in its inactive form.59, 60 However, several mutations have been reported, the
most important one being located at the residue Q61, which accounts for 86% of all N-Ras
mutations detected. To date, the Q61K, Q61R, Q61L, and Q61E mutational status have been
inventoried. This results in a dramatic decrease in GTP hydrolysis of up to tenfold compared
to wild-type Ras.59 In addition to Q61, two alternative residues are affected by mutations, that
is, G12 and G13.61 Since Ras is found in its mutant form in approximately 20% of melanoma
cases, its targeting has been seriously considered. Nevertheless, it appears that direct N-Ras
targeting is still an unmet challenge.60 Moreover, the approval of the first B-Raf inhibitor in
2011 brought most attention on B-Raf protein targeting. As a consequence, no marketed Ras
inhibitor is available to date, for the treatment of metastatic melanoma.
C. Raf
The Raf protein is located downstream from the Ras protein and turned out to be the cor-
nerstone in metastatic melanoma therapy since 2011. Human Raf has three isoforms; A-Raf,
B-Raf, and C-Raf. Of note, B-Raf presents the highest basal kinase activity62 and is considered
as the most potent activator of the downstream effectors MEK1 and MEK2,63 thus making it a
highly relevant target.12 A focus on the B-Raf isoform structure revealed a kinase domain com-
posed of two lobes separated by a catalytic cleft.62 In its inactive form, the lobes are close one
to another and stabilized by means of hydrophobic interactions. This inactive conformation,
called “DFG-out form,” hampers access to the catalytic cleft. To switch into its active form,
B-Raf must be phosphorylated by the Ras protein at two key residues (T599 and S602) that
are located in the B-Raf activation segment. The phosphorylation results in the destabilization
of the hydrophobic interactions between the two lobes. As a consequence, the protein flips to
its active conformation and the catalytic cleft becomes accessible.63–65 It is noteworthy that
wild-type Raf is able to activate MEK1 and MEK2 alone, but mostly through the formation of
homo- and heterodimers (e.g., B-Raf/B-Raf, C-Raf/C-Raf, B-Raf/C-Raf, and so on).64, 66–68
However, the mutations of the B-Raf isoform are frequent and result in its constitutive acti-
vation. This is the consequence of the mutation of key residues involved in the hydrophobic
interactions between the small and the large lobe of the protein.62, 69 A genomic analysis of the
B-Raf gene from 15 cancer cell lines highlighted three single-base substitutions. These consist in
one mutation in exon-11 (G1403C) and two within exon-15 (C1786G and the most important
one in melanoma, T1796A). Further analysis of 34 melanoma cell lines revealed 19 exon-15
mutations (56%), all of T1796A type. To date, approximately 70 mutated forms of B-Raf have
been characterized in cancers and the B-RafV600E mutant is highly predominant in melanoma
(70–90% of all B-RafV600 mutations).64 In this mutation, the valine residue at position 600 is
replaced by a glutamic acid. The negatively charged backbone of the glutamic acid mimics
the phosphate moiety given by Ras to activate B-Raf. As a consequence, B-Raf is locked in

106 r MILLET ET AL.
its active conformation, and exhibits an increased kinase activity.69 Strikingly, the B-RafV600E
mutant is 500-fold more active than wild-type B-Raf and is able to activate the downstream
effectors MEK1 and MEK2 alone.60, 63
D. Mek
The MEK protein is the third member of the MAPK/ERK pathway and is downstream Raf
proteins. It exists as two close isomers MEK 1 and MEK 2 (MAPKK). MEK1 and MEK2 share
86% identical residues in the catalytic domain and are equally able to phosphorylate ERK1/2
substrates.56, 70 They phosphorylate Thr202 and Tyr204 residues within the activation loop of
ERK1/2. To date, these are the only known substrates of MEK.71 Contrary to B-Raf, mutated
forms of MEK are found in low occurrence among human tumors. As MEK is positioned
downstream of Raf and has a narrow substrate tolerance, it has become a valuable target for
the treatment of the MAPK/ERK mutated forms of melanoma (e.g., N-Ras of B-Raf).
4. THE RISE OF TARGETED THERAPIES—PART 1: B-RAF INHIBITORS
Following these findings on the MAPK/ERK pathway mutations and their implication in the
melanoma, several drug discovery programs were undertaken. This resulted in the commercial-
ization and use of the first B-Raf inhibitor in the treatment of melanoma in 2011.
A. Vemurafenib (PLX-4032), 2011

Vemurafenib is the first marketed B-Raf inhibitor for the treatment of the metastatic melanoma.
1. Synthesis
Vemurafenib preparation was first reported following a convenient six-step synthesis.72 Herein,
a more suitable decagram-scale convergent synthesis starting from the commercially available
5-bromo-7-azaindole 8 is described. The synthesis started with a Suzuki coupling between 8
and 4-chlorophenylboronic acid 9 to yield bisaryl 10 (81%, Scheme 3).73
2,4-Difluoroaniline (11) was reacted with propylsulfonyl chloride to furnish 12 in a quan-
titative yield and subsequently formylated with N-formylmorpholine.74 Then, intermediates 10
and 13 underwent coupling under basic conditions (e.g., KOH), followed by treatment with
hydrobromic acid. Finally, the hydroxyl derivative was oxidized to the corresponding ketone
using 2,3-dichloro-4,5-dicyanoquinone (DDQ) yielding vemurafenib (14) (45% over three steps,
18.6% overall yield). Of note, 78.7 g of vemurafenib were obtained using this decagram-scale
synthetic pathway.
2. Drug Discovery
Vemurafenib was discovered following a fragment-based drug discovery. This approach consists
of setting up and screening a library of small and low affinity scaffolds. These are then combined
and grown up to access to a potential lead structure. Initially, a library of 20,000 compounds
was screened against a panel of selected kinases to discover a new potent B-Raf inhibitor
(Fig. 4).75 Compound properties were based on the following parameters: (i) low molecular
mass (150–350 Da), (ii) less than eight H-bond donors and acceptors, (iii) few rotatable bonds,
and (iv) relatively high aqueous solubility. The screening procedure was initiated at 200 μM
against selected kinases; Pim-1, p38, FGFR1, and CSK.74 Pim-1 and FGFR-1 provided robust
protein models, easy to crystallize with small and low-potent ligands, since B-Raf crystallization

Scheme 3. Vemurafenib decagram synthesis route.
Figure 4. Summary of the structure-based drug discovery program conducted to find vemurafenib, leading to
the discovery of the initial 3-aniline-7-azaindole (15) scaffold.
conditions were still under investigation.74 From the initial screening, 238 compounds were
suitable for co-crystallization. The selection was achieved using an upper limit of inhibition
set at 30% against at least three of the selected kinases. Among these 238 compounds, only a
hundred were effectively co-crystallized with Pim-1. This drew attention to the 3-substituted
7-azaindole scaffold (3-aniline-7-azaindole, 15). The 7-azaindole framework showed promising
properties with two H-bonds oriented toward the hinge pocket of the kinase. In addition,
putative substitution sites in positions 2, 3, 4, 5, and 6 were attractive. Due to low potency against
Pim-1 and multiple binding modes, a small set of additional derivatizations were performed on
scaffold 15. The replacement of the aniline substituent with a 3-methoxybenzyl moiety led to
the discovery of 16 harboring a 50-fold improved potency toward FGFR1. Compound 16 was
interesting since it was linked to the kinase pocket in a single binding mode, and presented an
increased number of binding interactions.
108 r MILLET ET AL.
Scheme 4. Hit-to-lead optimization of 16 toward vemurafenib (14) discovery.
Once the B-Raf protein was successfully crystallized, the optimization of 16 progressed
rapidly. Following an iterative structure-activity based lead improvement strategy (Scheme 4),
many efforts were focused on selective modifications of positions 3, 4, and 5. The diversifi-
cation of these positions resulted in the discovery of scaffold 17 that showed a crucial 2,6-
difluorophenyl moiety, substituted with a sulfonamide group in position 3. Subsequently, the
methylene linker in position 3 was replaced by a ketone linker that improved potency. Co-crystal
structures of 17 in B-RafV600E allowed further optimization of the sulfonamide substituent to
the optimal propyl group, thus leading to 18. The propyl group brought the desired B-RafV600E
selectivity among various kinases including B-Raf wild-type (IC50 of 13 and 160 nM, re-
spectively). A final optimization on the 5-position of the 7-azaindole was made by linking a
4-chlorophenyl moiety, which leads to vemurafenib (14), with improved pharmacokinetics. Of
note, this was the first successful approach of the fragment based drug discovery strategy.
3. Binding Mode
Interestingly, the co-crystallization of vemurafenib in a complex with B-RafV600E shows a
dimeric structure with both protomers in their active form (Fig. 5A).73 Vemurafenib binds
the ATP-binding site of only one of the protomers. Unlike other B-RafV600E inhibitors, such
interaction results in a shift of the regulatory αC-helix.75 Closer inspection of the docking of
vemurafenib in the B-Raf dimer revealed the following interactions. First of all, the arylsul-
fonamide moiety interacts with the backbone amide of Asp594 and Phe595 and the propyl
chain dives into a small hydrophobic pocket unique to the Raf family (Fig. 5B). Second, the
7-azaindole core occupies the hydrophobic cleft close to the hinge region of B-RafV600E . It is

Figure 5. (A) Crystal structure of vemurafenib with the B-RafV600E dimer (PDB structure 3OG7). Vemurafenib-
bound protomer (gray-blue) and free protomer (cyan). The bound protomer is overlaid with the crystal stucture
of another B-Raf inhibitor, BAY-43-9006 (green). (B) Structure of vemurafenib (green) bound in the ATP pocket.
The red dotted lines indicate H-bonds.
tightly confined in the adenine-binding region of the ATP-pocket and surrounded by several
residues such as Cys532, Trp531, Thr529, Leu514, and Ala481. The nitrogen 7 accepts an
H-bond from the backbone of Cys532 while nitrogen 1 donates an H-bond to Gln530. Third,
the ketone is involved in water bridged interactions. Finally, the difluorophenyl moiety settles
into a hydrophobic pocket containing eight residues (Ala481, Val482, Lys483, Val471, Ile527,
Thr529, Leu514, and Phe583).
4. Efficacy and Clinical Trials

In in vitro enzymatic assays, vemurafenib inhibits B-RafV600E with an IC50 of 31 nM. As a
comparison, it needs up to 100 nM to reach the enzymatic IC50 against B-Raf wild type.73
It presents good selectivity against a large panel of kinases (data not shown). The in cellulo
activity of vemurafenib is highly dependent on the mutational status of tested cell lines. It
reaches excellent in vitro growth inhibition values against six melanoma and breast cell lines
harboring the B-RafV600E mutation (Table I).76 Vemurafenib also retains good GI50 values
against alternative B-RafV600 mutational status (V600R, V600D, K601E). However, its activity
dramatically drops in the six cell lines (melanoma, lung and pancreatic) harboring a wild-type
form of B-Raf.

110 r MILLET ET AL.
Table I. In Vitro Inhibitory Activity of the Vemurafenib Against Mutated and WT B-Raf Cell Lines
Cell lines B-Raf status Ras status GC50 (μM)a
MALME-3M <0.02
Colo829 <0.03
Colo38 V600E <0.05
A375 <0.06
SK-Mel-28 WT <0.10
WM1341D Melanoma V600R <0.07
WM2664 V600D <0.20
WM239A V600D <0.30
WM3152 V600K <0.90
CHL-1 WT >10
SK-Mel-2 WT Q61R >10
H1666 Lung G466V WT >10
H460 WT Q61H >10
MDA-MB-435 Breast V600E WT <0.04
BxPC3 Pancreatic WT WT >10
MiaPaCa-2 G12C >10
Panc-1 G12D >10
a
Values extracted from a bar chart in 76.
Clear benefits have been demonstrated in a phase III clinical trial.36 A total of 675 ran-
domized patients were enrolled for this study; all harbored the B-RafV600E mutation and a
melanoma form that had not been treated previous. The aim of the study was to evaluate the
safety and the efficiency of vemurafenib as a monotherapy compared with DTIC for patients
with unresectable stage IIIc or IV melanoma. The results of this clinical trial showed more than
50% overall response rate for the group of patients treated with vemurafenib (5% with DTIC)
and a median progression-free survival (PFS) of approximately 7 months. The overall survival
(OS) at 6 months was 84% in vemurafenib group against 64% in the DTIC group. Moreover, the
median OS with vemurafenib was up to 13.3 months versus 10 months with DTIC.77 The most
commonly observed adverse effects during vemurafenib treatment are arthralgia, skin rash,
nausea, fatigue, photosensitivity, and, paradoxically, the development of small skin tumors
(squamous-cell carcinomas, keratoacanthomas).36, 78 These excellent results led to the approval
of vemurafenib in 2011 for the treatment of unresectable metastatic melanoma. Its first-line use
is restricted to the B-RafV600E mutated form of the disease.
B. Dabrafenib (GSK2118436), 2013

The approval of vemurafenib signified the beginning of a “gold rush” in the field of targeted
agents for melanoma. In this context, another B-Raf inhibitor, dabrafenib, was approved by the
FDA in 2013.
1. Synthesis
The synthesis of dabrafenib was originally described using a five-step synthetic pathway79
starting from the 2-fluoro-3-bromobenzoic acid (19, Scheme 5). The synthesis was initiated with
the esterification of the carboxylic acid function and the bromide function was subsequently
aminated through a palladium-catalyzed reaction to form 20. The resulting aniline intermediate
(20) reacted with the 2,6-difluorophenylsulfonyl chloride to yield the sulfonamide compound
21. Then, the lithiated 2-chloro-4-methylpyrimidine was condensed onto the ester function of

Scheme 5. Chemical route to the synthesis of dabrafenib.
21 to generate the desired ketone 22. Next, the thiazole core of 23 was formed by a Hantschz
reaction with 22 and the tert-butyl thioamide. Finally, dabrafenib 24 was obtained after the
conversion of the chloropyrimidine moiety into the corresponding aminopyrimidine in a 14.1%
overall yield.
2. Drug Design
The first hit (25) was discovered during an oncology program directed against multiple kinases
relevant to oncology.80 The prepared compounds were assayed using three in vitro biochemical
features: enzymatic activity against B-RafV600E , level of ERK phosphorylation (pERK), and
antiproliferative activity on SK-Mel-28 cells. Compound 25 displayed only modest inhibition of
ERK phosphorylation and cell proliferation (Scheme 6). Based on 25, three relevant structural
sections were identified as key SAR points; the head group and its amide linker, the imidazo[1,2-
a]pyridine core, and the N-methyltetrahydroisoquinoline tail. Varying from the amide to a
sulfonamide linker (26) resulted in a substantial decrease in the level of ERK phosphorylation.
However, the GI50 against SK-Mel-28 (1109 nM) was not enough. Efficient improvement of the
antiproliferative activity in cells was achieved by the replacement of the imidazo[1,2-a]pyridine
by the 1,3-thiazole ring. Thus, 27 reached a submicromolar activity against SK-Mel-28 cell
proliferation (GI50 = 287 nM) and kept strong inhibitory effects on both B-RafV600E and ERK
phosphorylation level. Subsequent modifications were carried out to improve both the potency
and the pharmacokinetic properties (PK) and led to the discovery of 28. Of note, the morpholine
ring was found to slightly improve the in vitro potency. Unfortunately, the intrinsic clearance
in rat miscrosomes was too high, while the distribution after oral administration was too low.
The stability of 28 was improved by the introduction of a fluorine atom at position 6 of
the phenyl ring, leading to compound 29. The latter exhibited a 70-fold increase of oral dose
area under the curve (AUC) in rat together with a reduced intrinsic clearance. Thus, 29 was

112 r MILLET ET AL.
Scheme 6. The hit-to-lead strategy developed for the optimization of dabrafenib (24) starting with the hit com-
pound 25.
Scheme 7. Metabolites identification of 30.
selected for further examination as a potential drug candidate. Unfortunately, its evaluation in
nonrodent species (dog and monkey) revealed very high clearance and poor bioavailability.79
A small subset of derivatives was synthesized to improve pharmacokinetic parameters. Hence,
dabrafenib (24), bearing a tert-butyl group and an unsubstituted aminopyrimidine ring, was
discovered. It displayed strong improvement in PK parameters (clearance and bioavailability)
in dog compared with 29. In order to understand the dramatic PK differences (high clearance
and poor oral exposure) between 30 and dabrafenib, a study was initiated. This revealed the
structures of 31 and 32 as major metabolites of 30 (Scheme 7).79 However, no biological activities
were provided by the authors.
3. Binding Mode
Dabrafenib is a reversible, ATP-competitive inhibitor of B-RafV600E .16 Its mode of action is
similar to vemurafenib. It binds to the ATP-pocket through hinge interactions between the one
of the nitrogen of the pyrimidine ring and the Cys532 residue (Fig. 6A).81 A structural overlay
of B-Raf bound with dabrafenib and vemurafenib indicates close protein conformation, and

Figure 6. (A) Co-crystal structure of dabrafenib (blue sticks) binds in the ATP-pocket of the B-RafV600E (PDB
file 4XV2). The red dotted lines indicate the H-bonds. (B) Overlay of vemurafenib (orange) and dabrafenib (gray)
bound B-RafV600E protein (PDB files: 4XV2 and 3OG7). (C) Overlay of dabrafenib (blue sticks) and vemurafenib
(orange sticks) in the ATP-pocket of B-RafV660E (PDB files: 4XV2 and 3OG7).
a comparable α-helix shift (Fig. 6B). Closer inspection of the two inhibitors overlaid in their
respective ATP-pocket reveals similar interactions with Asp594, Phe595, and Cys532 residues
(Fig. 6C).80 The difluorophenyl group of dabrafenib binds to the same lipophilic back pocket
than the propyl group in vemurafenib.82

Dabrafenib displays high in vitro potency in both enzymatic and cellular assays.79, 83 Its en-
zymatic IC50 against B-RafV600E , B-Raf wild type, and C-Raf wild type are in the same range
(0.7, 5.2, and 6.3 nM, respectively). It is also capable of inhibiting ERK phosphorylation with

114 r MILLET ET AL.
Table II. In Vitro Efficacy of the Dabrafenib in Cell Lines Harboring Various B-Raf Mutational Status
Cell lines B-Raf status Ras status GI50 (nM)a,b
MALME-3M 1
A375 4
SK-Mel-28 V600E 3
UACC-62 WT 1
M14 Melanoma 2
WM-115 V600D 5
YUMAC V600K 5
MDA-MB-468 Breast WT WT >10,000
BxPC3 Pancreatic WT WT >10,000
MiaPaCa >10,000
a
The growth inhibition values of dabrafenib are available against 195 cell lines. See 83 for more
information.
b
72-hr incubation.
an EC50 of 4 nM. At the cellular level, dabrafenib selectively inhibits the growth of B-RafV600
mutant cell lines. It blocks the growth of a panel of seven melanoma cell lines with B-Raf muta-
tions, V600E, V600D, and V600K (Table II). However, its activity dramatically decreases when
B-Raf is found under its wild-type form, thus demonstrating its high selectivity toward mutated
forms of B-Raf in cells. Of note, dabrafenib has no activity against breast and pancreatic cell
lines expressing a wild-type form of B-Raf.
Encouraged by the results of successful preclinical and phase I/II clinical trial studies,
a phase III clinical trial was initiated in patients with previously untreated, metastatic, or
unresectable B-RafV600E type melanoma37 . A total of 250 patients were enrolled and randomly
assigned to receive either dabrafenib or DTIC. Regarding tumor response, patients treated
with dabrafenib showed an RR of 50% (6% with DTIC) and a median PFS of 5.1 months
(2.7 months with DTIC). Updated survival data reported an improvement in the median OS
of 18 months while it was over 15 months for DTIC.84 While dabrafenib and vemurafenib
displayed similar efficacy in clinical trials, significant differences exists in terms of toxicity.85, 86
Even though these drugs have not been directly compared in a clinical trial, cutaneous toxicity
seems to occur less frequently with dabrafenib than vemurafenib. In addition, other side effects
such as arthralgia and fatigue also tend to be less frequent in patients undergoing dabrafenib
therapy.
These findings resulted in the approval of dabrafenib for the first-line treatment of unre-
sectable metastatic melanoma. It is use is limited to the B-RafV600E mutant form of melanoma.
C. Encorafenib (LGX818)
Encorafenib is a novel orally available and selective B-RafV600E inhibitor.87 To date, it is still un-
der clinical investigations and has not been approved by the FDA. It was developed to maximize
the response level and duration in patients by capitalizing on advantageous pharmacological
and activity properties.88
1. Synthesis
The synthesis of encorafenib was reported in 2011 using a 14-step gram-scale chemical path-
way (Scheme 8).89 First of all, isopropylhydrazine hydrochloride (33) reacted with benzalde-
hyde to form the imine intermediate 34 that underwent subsequent functionalization using

Scheme 8. 14-step chemical pathway for encorafenib (44) synthesis
(ethoxyethylidine)malononitrile to furnish 35. The pyrazole core of 36 was formed after heating
in acidic conditions. The nitrile function of 36 was converted into an acetyl group using methyl-
lithium, yielding 37. Then, the amino group was converted into diazonium and substituted
with iodide. The reaction of 38 with Bredereck’s reagent led to the formation of the derivative
39. Then, conversion of 39 into the corresponding chloropyrimidine 40 was accomplished in
three steps: first, 39 was cyclized with guanidine carbonate to form the 2-aminopyrimidine ring.
Second, the aminopyrimidine was substituted with a hydroxyl group. Third, chlorination in the
presence of phosphorus oxychloride yielded 40. Next, the 2-chloropyrimidine derivative 40 un-
derwent a nucleophilic aromatic substitution with (S)-methyl-1-aminopropan-2-ylcarbamate
hydrochloride, leading to 41.
The resulting compound (41) was engaged in a Suzuki cross-coupling reaction with 42 to
afford the derivative 43. Finally, classical acidic conditions were used to selectively remove the
BOC-protecting group from the aniline; the resulting free amine was mesylated prior to basic
treatment with sodium hydroxide to afford encorafenib (44). The chemical pathway depicted
herein corresponds to the pre-industrial-scale synthesis—483 g of 43 was prepared in a single
batch. It was described together with a laboratory-scale synthesis. Unfortunately, the authors
provided no yields.
The laboratory synthesis, described by Hueng,89 is very similar to the multigram-scale syn-
thesis described above, but there are some differences. Notably, the hazardous and pyrophoric
reagents have been substituted ongoing from laboratory scale to larger scale. For example, in
the second step of the synthesis, n-BuLi was replaced by DMAP and the methyllithium was
preferred over methylmagnesium bromide in step 4. The use of sodium nitrite, highly explosive,
to form the transient diazonium species in step 5 was avoided and the employment of isoamyl
nitrite, a more convenient organic nitrite reagent was chosen.
2. Binding Mode
To date, there is no available information concerning the drug-discovery program and the bind-
ing mode of encorafenib. Nonetheless, encorafenib, together with vemurafenib and dabrafenib,
might bind to B-RafV600E in a very similar fashion and implies similar conformational changes.81

Encorafenib was evaluated in vitro; it and inhibits ERK phosphorylation (EC50 ) at 3 nM and
shows an EC50 of 0.3 nM against B-RafV600E . It exhibits an IC50 as low as 4 nM in cell viability

116 r MILLET ET AL.
assays against A375 melanoma cells.89, 90 Like other B-RafV600E inhibitors, encorafenib did not
display any potency over more than 400 B-Raf wild-type cell lines (data not shown).88 Thus, a
phase I clinical trial was initiated. This was conducted with 54 patients harboring stage III or
IV unresectable melanoma91 ; 26 patients were not previously treated with B-Raf inhibitors and
28 patients were previously treated with B-Raf inhibitors. The RR reached 58% in patients not
previously treated, while it dropped to 11% in patients previously treated with B-Raf inhibitors.
The study provided a median PFS of 7.1 months and 1.9 months for non-previously treated and
pretreated patients, respectively. To date, a phase III clinical trial is ongoing (NCT01909453)
to assess the efficacy of encorafenib in B-Raf mutated melanoma; as a monotherapy and in
combination with the MEK inhibitor, binimetinib (vide infra). Preliminary results revealed cu-
taneous events as common side effects, such as palmar-plantar erythrodysesthesia syndrome,
hyperkeratosis, and keratosis pilaris. Several other disorders were noticed such as gastrointesti-
nal and musculoskeletal disorders, pain, fatigue, pyrexia, and photosensitivity.91 Nevertheless,
these initial data and the occurrence of side effects suggest that encorafenib may have a more
favorable toxicity profile compared with dabrafenib and vemurafenib.85
The availability of B-Raf inhibitors has allowed considerable improvements in the treatment
of metastatic melanoma. However, these inhibitors failed in the retention of a durable response
and are often subject to resistance mechanisms. The occurrence of such resistance will be
discussed later in the review.
5. THE RISE OF TARGETED THERAPIES—PART 2: MEK INHIBITORS
In parallel to the development of B-Raf inhibitors, numerous studies were focused on the
discovery of novel MEK inhibitors. This is the second targeted molecular biomarker within
melanoma.92 Several inhibitors were described in 2004 and have been co-crystallized with
MEK.71 The crystal structures revealed that MEK possesses a unique allosteric pocket ad-
jacent to, but separated from, the ATP-binding pocket. Upon inhibitor binding within the
allosteric pocket, conformational changes induce the lock of unphosphorylated MEK 1/2
into a catalytically inactive state.70 Of note, during the development of MEK inhibitors, no
selectivity between MEK1 and MEK2 could be reached due to their high sequence similarity.
A. Trametinib (GSK1120212), 2013

Trametinib was the first orally bioavailable MEK1 and MEK2 inhibitor approved by the FDA
in 2013 as a single-agent therapy for the treatment of metastatic melanoma.
1. Synthesis
The first reported synthesis of trametinib was a milligram-scale synthesis93 following a ten-
step chemical procedure starting from 2-fluoro-4-iodophenylisocyanate (45, Scheme 9). The
synthesis started with its coupling with cyclopropylamine to from 46. Next, 46 reacted with
malonic acid to allow the formation of the pyrimidin-2,4,6-trione scaffold 47. Subsequent
chlorination in the presence of phosphorus oxychloride yields the chloride derivative 48. The
newly introduced chloride group was then substituted with methylamine to furnish 49. Then,
the reaction of 49 with diethyl malonate allows the formation of the pyrido[2,3-d]pyrimidine
ring 50. The hydroxy group was coupled with trifluoromethanesulfonyl anhydride to form the
trifluoromethylsulfonyl intermediate (51) before its substitution by 3-nitroaniline to afford 52.
Then, the pyrido[2,3-d]pyrimidine scaffold 52 undergoes a rearrangement in basic media to yield
the pyrido[3,4-d]pyrimidine core 53. Subsequently, the nitro group was reduced using sodium
Scheme 9. Laboratory-scale synthesis of trametinib (55).
thiosulfate and the resulting aniline 54 was acetylated in the presence of acetic anhydride to
form the title compound trametinib (55) in a 0.74% overall yield. This poor overall yield is
mainly due to on the critical chlorination reaction on step 3 (only 8% yield).
An improvement of the chemical route toward trametinib was reported by Kawasaki94
and allowed its preparation on a gram scale. A ten-step synthesis was described (Scheme 10)
starting from 2-fluoro-4-iodoaniline 56. The synthesis commenced with the conversion of the
amino function into an isocyanate group followed by its coupling with cyclopropylamine to
form urea derivative 46. Next, 46 was coupled with cyanoacetic acid to allow the formation of
57 which was subsequently cyclized in basic aqueous media (NaOH) to obtain 58. Subsequent
coupling with N,N-dimethylformamide dimethylacetal yielded the derivative 59. Then, 59 was
reduced using sodium borohydride and cyclized with the methylmalonic acid to form the
pyrido[2,3-d]pyrimidine ring 50. Next, the hydroxy group was tosylated and substituted with
N-(3-aminophenyl)acetamide in the presence of 2,6-lutidine to form compound 60. Finally,
the pyrido[2,3-d]pyrimidine scaffold 60 underwent rearrangement in the presence of sodium
methanoate to yield trametinib (55). This optimization of the laboratory-scale synthesis resulted
in a dramatic improvement in the overall yield, from 0.74 to 32.4%.
2. Drug Design
The discovery of trametinib was the result of high-throughput screening followed by a SAR-
based structure optimization (Scheme 11). The screening was initially focused on compounds
able to induce the expression of p15INK4b , an inhibitor of cyclin-dependant kinase (CDK) 4/6.
This screening led to the identification of 61, a pyrido[2,3-d]pyrimidine based compound bear-
ing three phenyl rings in positions 1, 3, and 5.93 It displayed good in vitro GI50 against colon
cancer (HT-29) and renal cancer cells (ACHN; 990 nM and 4800 nM, respectively). However,
the low aqueous solubility (ClogP 6.3) of 61 was crippling for a future drug candidate. Thus,
each phenyl ring was replaced with various small alkyl groups to improve the hydrophilic prop-
erties. Interestingly, phenyl groups in positions 1 and 5 could not be swapped as it induced a loss

118 r MILLET ET AL.
Scheme 10. Gram-scale trametinib (55) synthesis.
of potency. In sharp contrast, the substitution of the phenyl ring in position 3 with a cyclopropyl
moiety enhanced the hydrophilicity (ClogP 5.0) and delivered a fourfold increase in activity (62).
Subsequent modifications were focused on positions 2, 3, and 4 of the aniline ring to improve the
potency. Among several small substituents (e.g., methyl, halogen, methoxy, etc.), the 2-fluoro
and the 4-iodo groups led to the best cellular activities. Of note, the bulkiness of the substituent
on the 4-position is strongly correlated to the biological activity and a bulky group is worse for
activity. All these structural improvements led to the discovery of 63, which brought a 100-fold
improvement in inhibitory activity compared to 62 with GI50 of 13 nM (ACHN cells) and
1.5 nM (HT-29). Unfortunately, the pyrido[2,3-d]pyrimidine core was unstable under weakly
basic conditions. Stabilization of 63 was accomplished via its rearrangement into the pyrido[4,3-
d]pyrimidine core 64. The latter (64) displayed similar potency (GI50 of 1.7 and 25 nM against
HT-29 and ACHN, respectively) to that observed with 63. While this rearrangement solved
the stability problem without affecting the activity, it had a dramatic impact on the aque-
ous solubility (solubility in PBS < 0.2μM). Thus, the phenyl ring in position 1 was subse-
quently functionalized with a 3-acetamide group, leading to the discovery of the structure of
trametinib (55).
This last substitution restored a decent aqueous solubility (ClogP 5.0, solubility of
5.3 μM in PBS) and improved bioavailability. This goes along with an improved potency
against HT-29 and ACHN cells, making trametinib a good drug candidate. The latter is for-
mulated as a DMSO solvate, presenting the desirable solid properties for galenic formulation
and clinical tests.
3. Binding Mode
Trametinib targets, MEK1 and MEK2, were elucidated using mutual exclusivity studies between
trametinib and another well-known allosteric inhibitor of MEK, PD0325901.95 The results
Scheme 11. Drug-design strategy used from the hit 61 to the lead compound trametinib (55).
showed that trametinib and PD0325901 bind to the same MEK binding domain, an allosteric
pocket adjacent to the ATP-binding site of MEK. Trametinib exerts dual activity; it inhibits the
phosphorylation of ERK1/2 but also decreases the phosphorylation levels of MEK1/2. The
binding of trametinib causes morphological/conformational changes in MEK1/2 that inhibit
its phosphorylation on serine residue 217.95 To date, no crystal structure of trametinib bound
to MEK is available.

Trametinib is a very potent inhibitor of both MEK1 and MEK2 (IC50 = 0.7 and 0.9 nM,
respectively). It inhibits the phosphorylation of ERK1/2 in SK-Mel-28 and HCT116 cells
(IC50 of 0.8 and 1.8 nM, respectively). It has been tested against several cell lines harboring
various Raf and Ras mutational status (Table III).95 Trametinib efficiently inhibits the growth
of melanoma and colon cell lines presenting B-RafV600E mutations. It is also efficient in cells
with RasG12S and RasG13N mutations but is completely inactive when B-Raf and Ras are the
wild-type form.

120 r MILLET ET AL.
Table III. GI50 Values of Trametinib Against Several Cell Lines In Vitro
Cell lines Cancer type B-Raf status Ras status GI50 a (nM)
A375PF11 Melanoma V600E WT 2.3

SK-Mel-28 Melanoma V600E WT 4
Colo205 V600E WT 1.7
HT29 Colon V600E WT 0.6
HCT116 WT G13N 21.1
PC3 Prostate WT WT >5,000
BT474 Breast >5,000
a
The growth inhibition was measured at 72 hr.
A phase III clinical trial was initiated with 322 patients with metastatic and unresectable B-
RafV600E or B-RafV600K mutated melanoma96 ; patients that underwent any previous treatment
were excluded from the study. Trametinib was assessed as a monotherapy and compared with
DTIC. The results showed an RR up to 22% in the trametinib arm versus 8% in the DTIC arm.
Median PFS duration was 4.8 versus 1.5 months in the trametinib and DTIC arms, respectively.
The median OS of patients under trametinib or DTIC was 15.6 versus 11.3 months, respectively.
Of note, the study highlighted a lack of efficacy of trametinib in B-RafV600K patients. Common
associated side effects were rash, diarrhea, peripheral edema, fatigue, and dermatitis acneiform.
These results prompted the FDA to approve the use of trametinib as a single agent for the
treatment of the B-RafV600E and B-RafV600K forms of melanoma. It is not indicated for patients
previously treated with B-Raf inhibitors.
B. Cobimetinib (GDC-0973, XL518), 2015

In 2015, cobimetinib became the second MEK inhibitor to be approved by the FDA. To date,
it has only been approved in combination with vemurafenib for the treatment of metastatic
melanoma.
1. Synthesis
The preparation of cobimetinib was first achieved using a convergent synthesis (Scheme 12).
The first diphenylamine building block 67 was synthetized according to a previously reported
procedure.97 A single-step LDA promoted nucleophilic aromatic substitution of 65 by aniline
66 leads to the formation of 67. The second intermediate, a 3-hydroxyazetidine derivative (68),
was prepared according to the procedure described by Beak et al.98 followed by an enantiomeric
resolution.
The two intermediates were then coupled together, through an amide bond formation
between the carboxylic acid group of 67 and the amino group of 68, to afford 69. Finally, the
N-Boc protection on the resulting compound 69 was removed using aqueous HCl to form the
title compound, cobimetinib hydrochloride (70).
2. Drug Discovery
The discovery of cobimetinib is closely related to the study of a diphenyl amine series that
was described by Orhen et al.70 Indeed, compound 71 served as synthetic platform.99 In this
context, the first drawback to overcome was the metabolic instability of the hydroxamate ester,
which is rapidly converted to the corresponding carboxylic acid. Moreover, in early clinical
trials, 71 and its metabolites were suspected to induce adverse effects such as ataxia, confusion,
and syncope.100

Scheme 12. Convergent synthesis developed for cobimetinib (70).
Thus, the first efforts were focused on the replacement of the unstable hydroxamate ester
function of 71 (Scheme 13). Its carboxamide analog 72 demonstrated significantly reduced
inhibitory effects. Nevertheless, other acyclic and cyclic carboxamide analogues were screened
and led to the use of the azetidin-3-ol scaffold (73) as an improved inhibitor of both biochemical
MEK activity and cellular ERK phosphorylation. However, this molecule has an unfavorable
pharmacokinetic profile, with low oral exposure and bioavailability in rat, high clearance, and
low aqueous solubility (ClogP 4.3).
Several amino substituents were assayed in the position 3 of the azetidin-3-ol scaffold
to improve aqueous solubility. As a result, the aminomethylene group (74) led to a significant
eightfold improvement in in vitro MEK and ERK phosphorylation inhibition. This was accom-
panied by better oral exposure and bioavailability in rat compared with 73. The clearance was
significantly reduced (11 times lower), thus resulting in the dramatic improvement of the AUC
and oral half-life values (31 and 6 times higher, respectively). Unfortunately, due to its blood–
brain barrier permeability and the need for a lower in vivo efficient dose (> 30 mgkg−1 day−1 ),
74 was not selected as a preclinical candidate.
Next, to minimize the unfavorable properties of this series, subsequent structural modifi-
cations were conducted. Alkylation, either at the amine moiety (75) or at the methylene bridge
(76) were surveyed. In both cases, it resulted in IC50 values in the same range as 74. However, the
combination of these modifications led to the discovery of the (S)-piperidyl ring harbored by
cobimetinib (70). This motif brought the desired pharmacokinetic properties along with strong
inhibitory effects. It resulted in the reduction of efficient in vivo doses (0.6 mgkg−1 day−1 ) and
minimal blood–brain barrier penetration. Hence, cobimetinib was selected for further clinical
evaluation.
3. Binding Mode
Like trametinib, cobimetinib binds to MEK1/2 in its allosteric pocket next to the ATP binding
site (Fig. 7A).99, 101 A closer inspection of this binding pocket shows that the azetidine arm
122 r MILLET ET AL.
Scheme 13. SAR study leading to cobimetinib (70) starting with the hit 71.
of cobimetinib projects efficiently into the catalytic loop region (Fig. 7B). Presumably, the
conformational constraint provided by the piperidine ring brings the amine close to the catalytic
loop, hence allowing the NH group hydrogen bond with Asp190. It also forms additional
interactions with the γ -phosphate of bound ANP.a Of note, the 3-hydroxyl group interacts with
both Asp190 and γ -phosphate.
The carbonyl of the amide linker serves as an H-bond acceptor from Lys97. The dipheny-
lamide backbone of cobimetinib is bound similarly to previous MEK inhibitors.70 The mode of
action of such inhibitors is highly dependent on the formation of H-bond interactions between
the 4-fluorine atom and Ser212. Overall, the activation loop of MEK is partially blocked and
prevents its phosphorylation at Ser218/Ser222.

Cobimetinib displays both high in vitro and in cellulo efficacy.101 It inhibits MEK with a pi-
comolar affinity (Ki = 0.05 nM) and inhibits the proliferation of A375 cells at 5 nM. The
Ki drops to 262.5 nM with ATP-free MEK, underpinning the importance of its interac-
tions with the γ -phosphate of bound ATP. Cobimetinib demonstrates efficient growth inhi-
bition against a panel of six melanoma and colon cell lines harboring B-RafV600E mutation
(Table IV). Cobimetinib is also able to inhibit the proliferation of various cancer cell lines
(pancreatic, breast, and colon) with mutated RasG12C , RasG12D , or RasG13D and wild-type
B-Raf.
The study of cobimetinib efficacy in combination with vemurafenib was conducted in a
phase III clinical trial.102 This study enrolled 495 patients with unresectable stage III or IV
B-RafV600E melanoma; patients were randomly selected for treatment with cobimetinib and ve-
murafenib in combination or vemurafenib alone. The results demonstrated a 68% RR including
a ANP is an ATP-analogue, in which the oxygen bridging the beta to the gamma phosphate has been replaced by a
nitrogen atom: it is less prone to hydrolysis than ATP.

Figure 7. (A) Co-crystal structure of cobimetinib (green sticks) and phosphoaminophosphonic acid-adenylate
ester, an ATP analogue (ANP, orange sticks) with MEK (PDB file 4LMN). (B) Binding interactions of cobimetinib.
The red dotted lines indicate H-bond interactions (PDB file 4LMN).
25 complete responses (10%) for the combination therapy versus 45% RR for vemurafenib alone
(11 complete responses, 4%). The respective overall survival at 9 months was 81% for combi-
nation therapy versus 73% for vemurafenib alone. During this study, the median OS was not
reached. The combination of vemurafenib and cobimetinib was associated with similar side
effects compared with vemurafenib, although some side effects were observed at higher fre-
quency such as central serous retinopathy, gastrointestinal events (diarrhea, nausea, vomiting),
photosensitivity, increased aminotransferase and creatine kinase levels. However, these side
effects were defined as manageable considering the efficacy of the therapy. To date, cobimetinib
has only been approved in combination with vemurafenib for the treatment of unresectable,
B-RafV600E or B-RafV600K metastatic melanoma.
C. Binimetinib (MEK-162, ARRY-162)

Binimetinib (77) is a non-ATP-competitive allosteric inhibitor of MEK1/2 (Fig. 8). Its structure
is closely related to the first series of diphenylamine MEK inhibitors described earlier (71,
Scheme 13) except that one of the phenyl rings was replaced with a benzimidazole system.
Binimetinib is currently being investigated as a monotherapy in a phase II clinical trial.103 For
this study, 71 patients with unresectable stage III or IV metastatic melanoma were enrolled. The
central gain of this clinical trial was the evaluation of the efficacy of binimetinib (single dose) in
patients harboring B-RafV600 , and also the N-Ras mutation. Furthermore, previous treatment

124 r MILLET ET AL.
Table IV. In Vitro Potency of the Cobimetinib Against Several B-Raf and Ras Mutated Cell Lines
Cell lines Cancer type B-Raf status Ras status EC50 (μM)a
A375b
888Mel Melanoma
C32 V600E WT
MALME-3M <0.5
Colo205b
HT-29b Colon
HCT116 WT G13D
MiaPaCa2 Pancreatic WT G12C <0.8
PATU 8988T WT G12V >1.0
MDA-MB-231 Breast G464V G13D <0.2
a
Data summarized from the chart, Figure 1A in 101.
b
Most sensitive cell lines in response to cobimetinib treatment. EC50 < 0.1 ␮M.
Figure 8. Structure of the MEK/1/2 in early clinical development.
(including chemotherapy, immunotherapy, or with B-Raf inhibitors but not MEK inhibitors)
was permitted. The RR appeared to be low (10% for N-Ras arm and 5% for B-Raf arm). The
median PFS was 3.7 months for patients with N-Ras mutated melanoma and 3.6 months for
patients with B-Raf mutated melanoma. These moderate results compared to trametinib can be
explained by the recruitment of previously treated patients. However, this is the first clinical trial
evaluating a drug that showed clinical activity in N-Ras mutated patients. Overall, binimetinib
was well tolerated and common side effects were skin- and gastrointestinal-related, as well as
fluid retention.
These findings justified additional phase III clinical trials. The first one is assessing binime-
tinib in monotherapy versus DTIC in 393 patients harboring N-Ras mutated, stage III or IV
unresectable melanoma (NCT01763164). The second study is evaluating the use of binime-
tinib in combination with encorafenib versus encorafenib and vemurafenib monotherapies in
a cohort composed of 900 patients with the B-RafV600 mutation (NCT01909453).
D. Other MEK Inhibitors

Additional MEK inhibitors are currently under early clinical investigation against metastatic
melanoma.71 Notably, pimasertib (78, AS703026 or MSC1936369B) is under investigation as a
monotherapy in N-Ras mutant, locally advanced or metastatic melanomas (Fig. 8). However,
the results of the phase II clinical trial (NCT01693068) are not yet available.

6. IMMUNOTHERAPIES
The revolution brought about by the approval of the first antimelanoma targeted therapy in
2011 was accompanied with the approval of a new generation of immunotherapies. Notably,
the immunotherapies allow the handling of B-Raf wild-type form of melanomas. So far, three
monoclonal antibodies targeting immune system receptors CTLA-4 and PD-1 have been ap-
proved for the treatment of metastatic melanoma. Ipilimumab, an anti-CTLA-4, was approved
in 2011 and the anti-PD-1 nivolumab and pembrolizumab were subsequently approved in 2014.
A. Ipilimumab, 2011
Ipilimumab is a fully human monoclonal antibody that targets cytotoxic T lymphocyte antigen-
4 (CTLA-4). The CTLA-4 protein is a complex mediator of immune homeostasis and tends
to negatively regulate the T-cell response.104, 105 The discovery of ipilimumab is closely related
to the understanding of the role of CTLA-4 in the immune system. Indeed, the blockade of
CTLA-4 in several murine tumor models attenuates the tumor growth.106 Moreover, CTLA-4
knock-out gene mice enhances the level of activated T cells.107, 108 So far, the CTLA-4 blockade
by ipilimumab demonstrates an improvement in the anti-tumor T-cell response.109 To obtain
further insights about the implication of CTLA-4 in immune response and melanoma, the
reader may refer to the publications and reviews in the references 110, 111 .
1. Efficacy
Two pivotal phase III clinical trial studies have demonstrated the efficacy of ipilimumab in the
treatment of metastatic melanoma. The first one compared ipilimumab alone, gp100 (a cancer
vaccine) alone, and the combination of ipilimumab and gp100 (Table V).112 676 patients with
unresectable stage III or IV metastatic melanoma were enrolled in this study. Previous treatment
with antineoplastic agents or IL-2 was permitted. The median overall survival was 10.0 months
for the combination and 10.1 months with ipilimumab alone. Analyses of the OS at 24 months
showed 21.6 and 23.5% in the combination and ipilimumab-alone groups, respectively. Finally,
the RR was 10.9% for ipilimumab alone compared with 5.7% for the combination. The results
with gp100 alone were unsatisfactory. The side effects were consistent with those enclosed in
previous phase II clinical trials,113, 114 the main ones being fatigue, diarrhea, pruritus, rash, and
colitis. Immune system related side effects were consistently observed during the treatment:
immune-mediated enterocolitis, hepatitis, dermatitis, or endocrinopathies.109
The second phase III clinical trial compared the efficacy of ipilimumab in combination with
DTIC versus DTIC alone (Table V).115 A total of 502 patients who had not received treatment
previously with stage III or IV metastatic melanoma were enrolled. The results showed an
overall RR of 15.2% for the combination therapy versus 10.3% for DTIC alone. The OS were
compared at 1, 2, and 3 years and respective data for the combination were 47.3, 28.5, and
20.8% compared with 36.3, 17.9, and 12.2% for DTIC alone. The significant improvement in
survival rate demonstrated in this study led to the approval of ipilimumab for the treatment
of unresectable metastatic melanoma. Notably, it is used as first-line treatment for the patients
with the B-Raf wild-type form of metastatic melanoma.
B. Nivolumab, 2014
The approval of nivolumab paved the way toward the second generation of monoclonal anti-
bodies used in the treatment of metastatic melanoma. It is a fully human monoclonal antibody
IgG4 directed against PD-1. The programmed death-1 (PD-1) protein is, like CTLA-4, a T-cell
126
Table V. Results of the Phase III Clinical Trial Concerning Ipilimumab

r MILLET ET AL.
Median PFS
Compound Clinical trial Study Parameters B-Raf status RR (%) (months) OS (%) CR (%)
Ipilimumab Phase 3 112 676 patients; M and Unknown 10.9: I 2.86: I 2 years 1.5: I
Hodi et al. C (gp100); previous 5.7: C 2.76: C 23.5: I

treatment with IL-2 21.6: C 0.2: C
or antineoplastic
agents authorized
Phase 3 115 502 patients; C Unknown 15.2: C / 2 years 1.6: C
Robert et al. (DTIC) vs. D; no 10.3: D 28.5: C
previous treatment 17.9: D
3 years 0.8: D
20.8: C
12.2: D
I, ipilimumab; D, DTIC; M, monotherapy; C, combination therapy; RR, response rate; OS, overall survival; CR, complete response.
co-inhibitor receptor and is activated through the binding with two known ligands, PD-L1 and
PD-L2.116, 117 Interestingly, PD-L1 is upregulated in many tumors, leading to reduced activity
of the immune system and the cytolytic activity of PD-1+ , CD4+ , and CD8+ T cells.118, 119
The inhibition of the PD-1/PD-L1 interaction potentiates the immune response in vitro and
showed preclinical antitumor activity.118, 119 For this reason, several anti-PD-1 antibodies have
been developed to fight cancer. For further details about PD-1 and PD-L1 implications in
cancer, the readers may refer to the reported publications.105, 116
1. Efficacy
The efficacy and safety of nivolumab have been investigated in two phase III clinical trials
(Table VI). The first involved nivolumab and ipilimumab alone and in combination, in a cohort
of 945 patients with stage III or IV unresectable melanoma.120, 121 No previous treatment was
permitted but all B-RafV600 mutational statuses were allowed. Patients were randomly assigned
in a 1:1:1 proportion with either mono or combination therapy. Median PFS of 11.5, 6.9, and
2.9 months were observed for combination, nivolumab alone, and ipilimumab alone, respec-
tively. A focus on the subgroup of patients harboring a wild-type form of B-Raf in the
nivolumab-plus-ipilimumab arm showed an 11.2 months PFS (v. 11.7 months in the B-Raf
mutated patients subgroup). This demonstrates equal efficiency of nivolumab, regardless of the
B-Raf mutational status. The RRs were 43.7% in the nivolumab group, 57.6% in the nivolumab-
plus-ipilimumab group, and 19.0% in the ipilimumab group. Of note, 28 patients (8.9%) and
36 patients (11.5%) demonstrated complete response with nivolumab alone or novilumab-plus-
ipilimumab therapy, respectively. The most commonly observed adverse effects were diarrhea,
fatigue, and pruritus. Moreover, like other immunotherapies, immune-related side effects were
also observed but considered to be manageable.
The second phase III clinical trial was initiated in patients with no B-Raf mutation and
previously untreated stage III or IV melanoma (Table VI).122 A total of 418 patients were
assigned randomly to receive nivolumab or DTIC. The median PFS reached 5.1 months in the
nivolumab group versus 2.2 months in the DTIC group. This was confirmed by an OS at 1 year
of 72.9% in the nivolumab group, compared with 42.1% in the DTIC arm. The RR appeared
to be high in the nivolumab group, up to 40.0% while only 13.9% of patients responded in
the dacarbazine group. Considering these results, the FDA approved the use of nivolumab
as a single agent for the treatment of patients who did not respond to other therapies (e.g.,
ipilimumab for B-Raf wild-type melanoma or B-Raf inhibitor for B-Raf mutated melanoma).
It is also indicated alone or in combination with ipilimumab for the treatment of patients with
B-Raf wild-type, unresectable metastatic melanoma.
C. Pembrolizumab, 2014
Pembrolizumab, another fully human monoclonal antibody, is the most recent anti-PD-1 drug
available for the treatment of metastatic melanoma. Its efficacy was evaluated in a phase II
clinical trial in 540 patients harboring unresectable stage III or IV melanoma in comparison
with DTIC (Table VI).123 This trial allowed the recruitment of previously treated patients
(with chemotherapies, immunotherapies, B-Raf, and MEK inhibitors) with confirmed disease
progression in response to ipilimumab therapy. Of note, the patients were administrated with
pembrolizumab at 2 or 10 mg and displayed a good overall RR of 21% and 25%, respectively. The
DTIC arm displayed only 4% RR. The PFS reached 5.4 and 5.8 months for the pembrolizumab
groups (2 and 10 mg/kg, respectively) versus 3.6 months for the DTIC group. Immune-mediated
side effects were scarce, while common side effects were fatigue, pruritus, nausea, diarrhea,
and rash.

128
Table VI. Summary of the Results Obtained During the Phase III Clinical Trials of the Anti-PD-1 Immunotherapies
Compound Clinical trial Study Parameters B-Raf status RR (%) Median PFS (months) OS (%) CR (%)
r MILLET ET AL.
Nivolumab Phase 3 120 945 patients, M and C All B-Raf 43.7: N 6.9: N 8.9: N
Larkin et al.(ipilimumab); no Status 57.6: C 11.5: C / 11.5: C
previous treatment 19.0: I 2.9: I 2.2: I
Phase 3 121 418 patients; M vs. D; B-Raf WT 40.0: N 5.1: N 1 year 7.6: N
Robert et al. no previous 13.9: D 2.2: D 72.9: N 1.0: D

treatment 42.1: D
Pembroli-zumab Phase 3 122 540 patients; M (two All B-Raf status 21: P (d1) 5.4: P (d1) 2: P (d1)
Ribas et al. doses) vs. D; 25: P (d2) 5.8: P (d2) / 3: P (d2)
previous treatment 4: D 3.6: D 0: D
with disease 1 year
progression
Phase 3 123 834 patients; M (two 33.7: P (d1) 5.5: P (d1) 74.1: P (d1) 5.0: P (d1)
Robert et al. doses) vs. ipilimu- 32.9: P (d2) 4.1: P (d2) 68.4: P (d2) 6.1: P (d2)
mab; one previous 11.9: I 2.8: I 58.2: I 1.4: I
treatment (no
immunotherapy)
RR, response rate; PFS, progression-free survival; OS, overall survival; CR, complete response; N, nivolumab; P, pembrolizumab; I, ipilimumab; D,
DTIC; M, monotherapy; C, combination; d1, dose 1; d2, dose 2.
Additional investigations were carried out in a phase III clinical trial (Table VI).124 The
criteria for inclusion in the study encompassed a broad scope of patients’ profile. Indeed, one
previous treatment was allowed (except immunotherapies) and all B-Rafmutational statuses
were accepted (but were determined prior to the treatment). Thus, 834 patients with stage III
or IV unresectable melanoma were recruited and randomly assigned (in a 1:1:1 proportion)
to receive either 10 mg/kg pembrolizumab every fortnight or every 3 weeks, or ipilimumab.
The RRs were similar in the two pembrolizumab groups (33.7% for fortnightly administration
and 32.9 % for administration every three weeks) and appeared to be higher compared with
ipilimumab (11.9%). The median PFS were 5.5 or 4.1 months in the pembrolizumab groups (2 or
3 weeks, respectively) versus 2.8 months in the ipilimumab group. In addition, severe treatment-
related adverse events (grades 3 to 5) appeared with a lower incidence in the pembrolizumab
groups (13.3 and 10.1%) than in the ipilimumab group (19.9%).
These key results were very promising and prompted the FDA to approve pembrolizumab in
2014 for the support of patients with unresectable metastatic melanoma with disease progression
following a previous treatment. The approval was extended in 2015 for the first line treatment
of patients with unresectable metastatic melanoma.
7. RESISTANCE, WHAT’S NEXT?
Notwithstanding extensive efforts to cure or circumvent the progression of the melanoma,

resistances are still a major issue. The occurrence of resistance, resulting from multiple biological
events, is a complex phenomenon dependent on the therapy that is received.
A. Resistance to Alkylating agents

Resistance to cytotoxic chemotherapies is well known and extensively documented in some key
references.125, 126 This is beyond the scope of this review and will not be discussed here.
B. Resistance to B-Raf inhibitors

As discussed, vemurafenib and dabrafenib represented a milestone in the treatment of metastatic
melanoma. They are able to extend the life span of patients diagnosed with metastatic
melanoma. In addition, dabrafenib permitted the reduction of some side effects and the treat-
ment of other B-RafV600 mutational statuses (V600K or V600D), compared with vemurafenib.
It also increased the median OS up to 18 months (vs. 13.3 months for vemurafenib). Nonethe-
less, despite quick response and tumor regression, most patients treated with dabrafenib or
vemurafenib relapse within 1 year.37, 127 Almost 70% of patients exhibit acquired resistance
while de novo resistance is rarer (10–20%).60, 128 In addition, acquired B-Raf inhibitor resis-
tance is a serious problem as the disease resurgence exhibits a stronger progression compared
with tumors that have not been previously treated.78 Even if mechanisms through which resis-
tance develops are not yet fully understood, several studies have attempted to elucidate them.
Two major phenomena implicated in the emergence of resistance following a B-Raf inhibitor
treatment have been validated so far.
The main mechanism of resistance consists of the reactivation of the MAPK/ERK
pathway52, 116 through the aberrant formation of Raf dimers (e.g., C-Raf/C-Raf or B-Raf/C-
Raf, etc.) since B-Raf inhibitors are only active against monomeric forms (Fig. 9).128 Strikingly,
this phenomenon does not rely on secondary mutations on the B-Raf protein.78 This dimeriza-
tion process may occur following various events.

130 r MILLET ET AL.
Figure 9. Major mechanisms of acquired resistance following B-Raf and/or MEK inhibitors treatments.
First, it may pass through the expression of aberrant spliced forms of B-Raf presenting no
Ras-binding domain. Consequently, they are able to dimerize in a Ras-independent fashion.129
Second, the N-Ras Q61K mutated form also induces the aberrant formation of Raf dimers.
This is frequently associated with the downregulation of the neurofibromin (NF1), a negative
regulator of Ras.130 Lastly, B-RafV600E inhibition may also lead to paradoxical activation of
the MEK/ERK cascade. Indeed, B-RafV600E inhibitors are able to bind to Raf dimers, caus-
ing the trans-activation of the other drug-free protomer and in fine the reactivation of the
pathway.78, 131–133 This phenomenon is commonly designed as the “paradoxical activation.”
Additional resistance mechanisms that imply the reactivation of MAPK/ERK pathway can
also arise through C-Raf or the action of cancer Osaka thyroid oncogene (COT)134 proteins.
These are able to activate MEK in a B-Raf-independent manner.135 Secondary mutations
of MEK 1 and MEK 2 proteins are also implicated in the downstream reactivation of the
MAPK/ERK pathway.60, 128
The second means by which melanoma cells acquire resistance relies on the over-activation
of alternative signaling pathways. Indeed, the concurrent activation of the PI3K/Akt pathway
by the upregulation of RTKs (e.g., insulin-like growth factor 1 receptor—IGF-1R or platelet-
derived growth factor receptor beta—PDGFRβ) leads to a loss of sensitivity to B-RafV600E
inhibitors.135 This is frequently associated with the loss of PTEN, which negatively regulates
PI3K.136, 137 In addition, several studies reported an increased level of PI3K pathway activation
following vemurafenib treatment in resistant cells.60, 75 As a result, the efficacy of treatments
will be diminished.

C. Resistance to MEK Inhibitors
The development of MEK inhibitors has strengthened the therapeutic arsenal for the treatment
of metastatic melanoma. Indeed, the approval of trametinib in 2013 allowed the support of
patients harboring B-RafV600E of B-RafV600K mutant forms of melanoma. The combination
of MEK and B-Raf inhibitors substantially increased the response duration. Unfortunately,
the development of MEK inhibitors such as trametinib and cobimetinib did not succeed in
stemming development of resistance but only delayed it.
This form of resistance is mainly due to the reactivation of the MAPK/ERK signaling
pathway due to MEK1/2 mutations. The latter are due to the prolonged exposure to MEK in-
hibitors. These mutations take place either on the binding site of the inhibitor (e.g., MEK1L115P ,
MEK1F129L , MEK2V211D , etc.), therefore preventing its binding, or may render MEK1/2 con-
stitutively active (e.g., MEK1F129L or MEK2Q60P ).138, 139 The amplification of B-RafV600E or
Ras oncogenes has also been observed in the development of resistance to MEK inhibitors by
increasing the pool of phosphorylated MEK1/2.140, 141 To a lesser extent, resistance to MEK
inhibitors may pass through either the overactivation of alternative pathways (e.g., PI3K/Akt,
STAT3, or Nf-Kb) or the loss of ERK1/2 negative feedback.142,b
D. Resistance to Immunotherapies
In parallel to the rise of B-Raf and MEK inhibitors, the support of wild-type B-Raf melanomas
was achieved using anti-CTLA-4 and anti-PD-1 immunotherapies. These allowed a broader
scope of treatment and are effective against both B-Raf mutated and wild-type melanomas.
In stark contrast, immunotherapies allow a more durable response after treatment compared
to targeted therapies. Indeed, the 1-year OS for anti-PD-1 (nivolumab and pembrolizumab)
reaches 75%. However, the response rate does not exceed 40–50% and the occurrence of resis-
tances (de novo or therapy-induced) is a major issue since no alternatives exist. The de novo
forms of resistance probably derive from several biological events that dampen T-cell activity
inside the tumor microenvironment. This mechanism relies on the expression of negatively-
regulatory sensors such as LAG-3, TIM-3, or BTLA.143 The overexpression of PD-L1 within
the surface of tumor cells, upon the production of IFN-gamma by T-cells, also limits T-cell effi-
cacy. Of note, the therapy-induced mechanisms of resistance are quite similar to those observed
in the de novo resistance. Detailed mechanisms underlying these two aspects of resistances are
available in the references.143
8. FUTURE PROSPECTS
As discussed, the mechanisms of resistance imply high levels of reactivation of the MAPK/ERK
pathway. Thus, several strategies have been considered to prevent the occurrence of such events.
A. Anti PD-L1 Antibodies

Another way to disrupt the PD-1/PD-L1 interaction, apart from the PD-1 targeting, relies on
the blockade of the PD-L1 ligand. It is the primary PD-1-ligand upregulated within tumor
environment. Hence, new anti-PD-L1 antibodies are currently under clinical evaluation and
show early promise. These studies concern MPDL3280A (NCT01375842),144 BMS-936559,145
b The ERK1/2 proteins are able to exert negative feedback on MAPK/ERK pathway to ensure a correct homeostasis.
They phosphorylate B-Raf, C-Raf, and MEK-1 to inhibit their kinase activity.

132 r MILLET ET AL.
Table VII. Clinical Evaluation of Combined Immunotherapies and Targeted Therapies
Antibodies Small molecules
Clinical trial number Name Target Name Target
NCT02357732 Nivolumab PD-1 Dabrafenib MEK

Trametinib MEK
NCT02027961 MEDI4736 PD-L1 Dabrafenib MEK
Trametinib MEK
NCT01656642 MPDL3280A or atezolizumab PD-L1 Vemurafenib B-Raf
Cobimetinib MEK
and MEDI4736 (NCT01693562)146 anti-PD-L1 antibodies. Of note, these antibodies are under
investigation in phase I or II clinical trial against melanoma and several other solid tumors.
B. Combination Therapies
The first valuable alternative that emerged to overcome the high reactivation level of the
MAPK/ERK pathway is the combination of B-Raf and MEK inhibitors. Indeed, preclin-
ical studies showed the additional MEK blocking could prevent the onset of resistance to
B-Raf inhibitors.147, 148 The dual B-Raf/MEK inhibition was thus able to kill resistant cancer
cells. Following these findings, the trametinib/dabrafenib combination therapy was recently
investigated.149 A phase I/II clinical trial in which 162 patients were enrolled, with no previous
B-Raf or MEK inhibitor treatment, were randomly administered with dabrafenib monother-
apy (150 mg) or dabrafenib in combination with trametinib (150 mg/2 mg). The RR observed
under the combination therapy was 76% and the 12 month PFS was 41%. Of note, nine patients
(5%) showed complete response. As a comparison, dabrafenib-only arm showed a 54% RR
(including four complete responses, 2%) and a 12 month PFS up to 9%. The administration
of a combination therapy caused more frequent and more severe pyrexia, chills, nausea, and
vomiting. However, a decrease in cutaneous side effects was observed. This is due to the mode
of action of trametinib that blocks the paradoxical activation of the MAPK/ERK pathway.149
Following these results, the FDA approved the use of the trametinib–dabrafenib combination
for the treatment of unresectable metastatic melanoma (B-RafV600E and B-RafV600K ) in 2015.
Of note, this was the first clinically used combined therapy for the treatment of the metastatic
melanoma. Since then, a second combination therapy was authorized, vemurafenib and cobime-
tinib combination (vide supra).102, 149 The related clinical studies demonstrated longer response
and a delayed development of resistance, compared with the corresponding monotherapies.
Nonetheless, resistance still develops after several months of treatment (average of 3.6 to
9.4 months).150, 151 In such cases, the MAPK pathway is once again reactivated.150–152,c
Nevertheless, another combination therapy binimetinib/encorafenib is under investigation
(NCT01909453).
Immunotherapy has also been investigated in combination with B-Raf and/or MEK
inhibitors. The first clinical trial assayed vemurafenib and ipilimumab in combination in a
small cohort of patients. However, due to early signs of hepatic toxicity, the study has been
canceled.153, 154 The emergence of the anti-PD-1 and anti-PD-L1 antibodies leads to the investi-
gation of several new clinical trials to assess immunotherapies/targeted therapies combination
but no results are available to date (Table VII).155
c The associated mechanisms of resistance have been deciphered and concern the production of spliced forms of
B-Raf. Subsequent mutations of MEK1C121S and more frequently MEK2C125S are also involved.

Figure 10. Structure of the Pan-Raf inhibitors in the pipelines.
C. Pan-Raf Inhibitors
The second option to circumvent the appearance of resistance is the development of Pan-Raf
inhibitors. A Pan-Raf inhibitor is able to simultaneously block the activity of the three Raf
isoforms; A, B, and C. The need to develop Pan-Raf inhibitors is based on key findings. First,
B-Raf inhibitor treatment is responsible for the paradoxical activation of MAPK pathway,
partly due to the other Raf isoforms. Second, the inhibition of C-Raf enhances MEK in-
hibitor activity.156 Recently, the compound LY3009120 (79) was proposed as Pan-Raf inhibitor
(Fig. 10). It exhibits IC50 values against purified B-Raf wild type, C-Raf wild type, and
B-RafV600E of 9.1, 15, and 5.8 nM, respectively.
Compound 79 also demonstrates in cellulo potency against B-Raf mutant, N-Ras mutant,
and vemurafenib-resistant cells. The co-crystal structure of 79 with B-Raf revealed that it
binds to both B-Raf protomers and thus inhibits MEK phosphorylation.156, 157 Other Pan-Raf
inhibitors are in the pipeline (Fig. 10). For instance, CCT196969 (80) and CCT241161 (81)158
show promising activities against various melanoma cell lines including vemurafenib-resistant
cells, but also dabrafenib- and trametinib-resistant cells. Finally, MLN-2480 (82) is another
Pan-Raf inhibitor currently undergoing a phase I clinical trial (NCT02014116).
D. ERK Inhibitors
In attempts to blockade the MAPK/ERK pathway, several ERK inhibitors have emerged.159
The recent interest in ERK1/2 targeting is based on two findings. First, as the dual block-
ade of B-Raf and MEK demonstrated promising results, the threefold inhibition of B-
Raf/MEK/ERK could lead to substantial benefits. Second, resistance to B-Raf or MEK
inhibitors frequently involves the reactivation of ERK signaling, thus justifying its targeting.150
To date, only a few ERK-inhibitors have been described and some of them are restricted to

134 r MILLET ET AL.
Figure 11. Structures of the ERK inhibitors.
Figure 12. Structures of PI3K inhibitors.
phase I clinical trial studies. Early results concerning SCH772984 (83) highlight the strong
inhibition of the proliferation of cells resistant to B-Raf and MEK inhibitors (Fig. 11).152
This ATP-competitive inhibitor binds to the unphosphorylated form of ERK and displays
an IC50 against ERK1 and ERK2 of 4 and 2 nM, respectively. Its binding induces an inhibition
of ERK1/2 kinase activity and prevents its phosphorylation by MEK.160, 161 Some other ERK
inhibitors are currently investigated in preclinical studies (VTX11e, 84 and (S)-14k, 85) while
more advanced compounds such as GDC-0994 (86) and ulixertinib (BVD-523, 87) are already
undergoing phase I clinical trials (Fig. 11).81, 159
E. Targeting of the PI3K/Akt Pathway

The lack of a durable response with the MAPK/ERK pathway blockade underlines the
need to explore alternative pathways. Thus, the PI3K/Akt pathway, the second most overreg-
ulated pathway in melanoma, has received much attention. Three potential drug targets were
identified within this pathway: PI3K, Akt, and mTor. Even though Akt and mTor have been
investigated using small molecule inhibitors such as the mTor inhibitor CCI-779,162 it is believed
that PI3K is the most promising druggable target.136 So far, the single PI3K pathway targeting
does not seem to be a pertinent option, whereas the dual inhibition of the MAPK/ERK and
PI3K/Akt pathways has already proved fruitful in early preclinical studies.163, 164
Thus, several clinical trials have evaluated the efficacy of the MAPK inhibitors combined
with PI3K or Akt inhibitors. In a phase I/II clinical trial (NCT01902173), dabrafenib and
trametinib have been assayed in combination with GSK2141795, an Akt inhibitor (88, Fig. 12).

Figure 13. Structure of the BiP inhibitor.
Another clinical study is currently evaluating encorafenib in combination with binimetinib or

BKM120 (89, NCT01820364). Vemurafenib is also being investigated with two PI3K inhibitors
(i.e., PX-866 (90), NCT01616199 and NCT01512251). Unfortunately, since the results of these
studies have not been published yet, it is too early to state on the clinical relevancy of such dual
inhibition.
F. Future Prospects
Aside from the blockade of the MAPK/ERK and PI3K/Akt pathways, alternative studies
are interested in the targeting of secondary signaling pathways involved at minor level in
melanoma. Of note, NF-κB has been implicated in the proliferation of melanoma through the
loss of senescence165 ; its blockade demonstrates in vivo reduction in melanoma tumor growth.
This goes along with a substantial increase in immune response following immunotherapy
(CTLA-4 or PD-1). However, the inhibition of cancer growth through the restauration of
senescence is still at a very early stage of development. Likewise, early interests in the JAK/STAT
pathway demonstrate interesting results.166 Its implication in melanoma is well known and could
represent an alternative to circumvent resistance.167 The inhibition of the signal transducer and
activator of transcription 3 (STAT3) protein succeeds in inhibiting cell growth in cells resistant
to vemurafenib.
Another promising strategy is the targeting of the Unfolded Protein Response (UPR)
pathway that appears as an emerging pathway to selectively target cancer cells. UPR is activated
in response to accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic
reticulum (ER). Malignant cells adapt their metabolism by activating the UPR to face the
high levels of unfolded proteins due to an abnormal high protein synthesis.168–170 Binding
immunoglobulin Protein (BiP) is a key enzyme in this process and has been found to be
overexpressed in many cancers.168–170 It plays a pivotal dual role of both UPR-sensor upstream
the pathway and also acts as a chaperone.171, 172 A BiP inhibitor (91, Fig. 13) has been recently
developed in our laboratories, which has shown significant in vitro and in vivo efficiencies on
melanoma cells, particularly against B-Raf inhibitors resistant cell lines.173 It is noteworthy
that 91 is able to block the growth of cells resistant to vemurafenib and dabrafenib, as well as
melanoma cells harboring several mutations (B-RafV600E , N-RasQ61K , B-Raf, and N-Ras wild
type). Nonetheless, these approaches might represent a promising approach to treat not only
B-Raf mutated patients, but also B-Raf wild-type patients and a variety of other cancers.
9. CONCLUSION
In summary, we have depicted the tremendous advances that have been achieved in the treatment
of metastatic melanoma. While initial therapies were limited to the use of antineoplastic agents,

136 r MILLET ET AL.
the emergence of immunotherapies and targeted therapies has revolutionized this field. It
has resulted in the dramatic improvement of patients’ care and life expectancy. Nonetheless,
significant barriers remain to be overcome, notably concerning the handling of resistance.
A large part of the current field of melanoma research is dedicated to the circumvention of
these mechanisms in response to treatment with targeted therapies. In this context, the blocking
of the MAPK/ERK pathway is still on the rise although this approach may only delay the
appearance of resistance. Therefore, a hypothetic triple inhibition of B-Raf, MEK and ERK
is under consideration, giving rise to the development of ERK inhibitors. In addition, we also
reviewed the emergence of new Pan-Raf inhibitors. These are more potent and less prone to
resistance than classical B-Raf inhibitors, and may provide an encouraging solution.
Bypassing the inefficiency of targeted therapies toward B-Raf wild-type forms of melanoma,
immunotherapies appeared as a breakthrough. They provide a fairly durable response and can
also serve as second line therapy for relapsed patients that underwent B-Raf inhibitor or anti-
CTLA-4 treatment. However, despite spectacular improvement in life expectancy, resistance
still arises, thus underpinning the need for new alternatives.
The future prospects turn toward the targeting of alternative pathways. Hence, the
PI3K/Akt pathway represents a real alternative in melanoma therapy and its inhibition will cer-
tainly be effective in a near future. The latest research has focused attention on the blockade of
minor pathways (e.g., NF-kB, JAK/STAT, or GRP78). These might be considered as medium-
term antimelanoma approaches, and could be beneficial for both mutant and wild-type B-Raf
melanoma forms.
ACKNOWLEDGMENTS
This work was supported by the French “Ministère de l’Education nationale, de l’Enseignement
supérieur et de la Recherche,” INSERM, CNRS, University of Nice Sophia-Antipolis (AM
grant), INSERM Transfert (COPOC grant), Canceropole PACA, and ARC (contract no. SFI
20121205378). INSERM U1065, team 1 is “équipe labelisée ligue 2010”. Dr. D. J. Nelson is
gratefully acknowledged for proof reading of the manuscript.
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Bertolotto C, Hofman P, Ballotti R, Benhida R, Rocchi S. New compounds triggering endoplasmic
reticulum stress exert anti-melanoma effects and overcome BRAF inhibitor resistance. Cancer Cell
2016;29:805–819.
Antoine Millet was born in Vienne (France) in 1990. He received his B.Sc. degree in organic
chemistry and his M.Sc. degree in organic and medicinal chemistry (with honors) from the
University of Montpellier 2 in 2011 and 2013, respectively. He then moved to the University of
Nice-Sophia-Antipolis to initiate a Ph.D. program under the supervision of the Dr. R. Benhida.
His research efforts are focused on the synthesis and structural optimization of new derivatives to
overcome resistant forms of malignant melanoma.
Anthony R. Martin was born in Sète (France) in 1984. He received his Ph.D. from the University of
Montpellier 2 in 2011 under the joined supervision of Prof. Michael Smietana and Dr. Jean-Jacques
Vasseur, working on the synthesis and self-assembly of borono(oligo)nucleotides. He then joined
Prof. Steven P. Nolan’s group (University of Saint-Andrews) as a postdoctoral research associate
to work on the coordination of late transition metals and their applications in homogeneous
catalysis. In 2013, he moved to the Institute of Chemistry of Nice as a lecturer in the group
of Dr R. Benhida and was appointed Chargé de Recherche—CNRS in 2014. His current research
interests focus on the design and preparation of new anticancer agents and encompass various
aspect of nucleic acid chemistry.
Cyril Ronco is an Assistant Professor at the Institute of Chemistry of Nice, France (ICN).
He received his Ph.D. degree in organic chemistry in the group of Prof. P.-Y. Renard at the
University of Rouen, France. He then joined the group of Prof. H.-D. Arndt at Jena University
(Germany) as a Humboldt postdoctoral fellow and worked on the total synthesis of complex
natural products. He worked after on medicinal chemistry projects at the Faculty of Pharmacy
of the University of Lille (France) in the group of Prof. B. Déprez. In 2014, he joined the group
of Dr. R. Benhida at the University of Nice and his research focuses on the development (design,
chemical derivatization, studies of mode of action) of bioactive molecules in the field of oncology.
148 r MILLET ET AL.
Stéphane Rocchi is currently director of research at INSERM, INSERM U1065 team 1, Bi-
ology and pathology of melanocyte, Centre Méditerranéen de Médecine Moléculaire (C3M).
He received his Ph.D. from University of Nice Sophia Antipolis, France (1998). After 2-year
postdoctoral position at Institut de Génétique et Biologie Moléculaire et Cellulaire (IGBMC),
he joined INSERM as Chargé de recherche in 2001. His current research is focused on molecular,
cellular, and clinical investigations of new antimelanoma drugs.
Rachid Benhida is currently director of research at CNRS, Co-Director of Nice Chemistry In-
stitute (ICN, Institut de Chimie de Nice, UMR CNRS-UNS 7272) and team leader (Bioactive
Molecules’ team at the ICN). He received his Ph.D. from University of Paris XI, Orsay, France
(1992). After three-year postdoctoral positions Hoechst/Roussel-Uclaf (1992–1993) and “In-
stitut Curie-Paris” (1993-1994), he joined the “Institut de Chimie des Substances Naturelles,”
ICSN, as ANRS postdoc award and as Chargé de recherche CNRS in the same year (1995). He
moved from ICSN to the University of Nice Sophia Antipolis in 2002 (LCMBA, UMR 6001). His
current research is focused on nucleic acids chemistry, the development of novel synthetic methods
and their applications in the synthesis of bioactive molecules. He is the co-founder and the current
Vice President of the Moroccan Society of Medicinal Chemistry (SMCT).
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Metastatic Melanoma: Insights Into the Evolution of the

Article in Medicinal Research Reviews · August 2016

Antoine Millet Anthony R Martin

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Cyril Ronco Stephane Rocchi

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Antoine Millet,1 Anthony R. Martin,1 # Cyril Ronco,1 # Stéphane Rocchi,2,3,4*

Published online in Wiley Online Library (wileyonlinelibrary.com).

# These authors contributed equally to this work.

Medicinal Research Reviews, 37, No. 1, 98–148, 2017

Medicinal Research Reviews DOI 10.1002/med

Figure 1. Chronology of the therapies approved to treat the metastatic melanoma.

2. THE USE OF FIRST-GENERATION THERAPIES

Medicinal Research Reviews DOI 10.1002/med

Medicinal Research Reviews DOI 10.1002/med

Scheme 2. Mode of action of the DTIC (2) and TMZ (6).

Medicinal Research Reviews DOI 10.1002/med

Figure 2. Developed structure of the fotemustine (7).

Medicinal Research Reviews DOI 10.1002/med

3. IMPLICATION OF MUTATED MAPK/ERK PATHWAY IN MELANOMA

A. MAPK/ERK Pathway Signaling

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Medicinal Research Reviews DOI 10.1002/med

4. THE RISE OF TARGETED THERAPIES—PART 1: B-RAF INHIBITORS

A. Vemurafenib (PLX-4032), 2011

Medicinal Research Reviews DOI 10.1002/med

Scheme 3. Vemurafenib decagram synthesis route.

Scheme 4. Hit-to-lead optimization of 16 toward vemurafenib (14) discovery.

Medicinal Research Reviews DOI 10.1002/med

4. Efficacy and Clinical Trials

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Cell lines B-Raf status Ras status GC50 (μM)a

B. Dabrafenib (GSK2118436), 2013

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Scheme 5. Chemical route to the synthesis of dabrafenib.

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Scheme 7. Metabolites identification of 30.

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4. Efficacy and Clinical Trials

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Cell lines B-Raf status Ras status GI50 (nM)a,b

Medicinal Research Reviews DOI 10.1002/med

Scheme 8. 14-step chemical pathway for encorafenib (44) synthesis

3. Efficacy and Clinical Trials

Medicinal Research Reviews DOI 10.1002/med

5. THE RISE OF TARGETED THERAPIES—PART 2: MEK INHIBITORS

A. Trametinib (GSK1120212), 2013

Scheme 9. Laboratory-scale synthesis of trametinib (55).

Medicinal Research Reviews DOI 10.1002/med

Scheme 10. Gram-scale trametinib (55) synthesis.

4. Efficacy and Clinical Trials

Medicinal Research Reviews DOI 10.1002/med

A375PF11 Melanoma V600E WT 2.3

B. Cobimetinib (GDC-0973, XL518), 2015

Medicinal Research Reviews DOI 10.1002/med

Scheme 12. Convergent synthesis developed for cobimetinib (70).

4. Efficacy and Clinical Trials

nitrogen atom: it is less prone to hydrolysis than ATP.